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https://f1000research.com/articles/11-234/v1	24 Feb 22	{ "type": "Research Article", "title": "The impact of clinical pharmacist-led health education on the disease course of non-alcoholic fatty liver disease patients: an interventional study", "authors": [ "Nehal Abou Seada", "Manal El Hamamsy", "Sarah Shaheen", "Reda Elwakil", "Alaa Barakat", "Azza El‐Sayed Mansy", "Manal El Hamamsy", "Sarah Shaheen", "Reda Elwakil", "Alaa Barakat", "Azza El‐Sayed Mansy" ], "abstract": "Background: A multitude of health care professionals, including pharmacists, are needed in managing a complex and silent disease like non-alcoholic fatty liver disease (NAFLD) associated with metabolic syndrome (MetS). These health care professionals can increase patients’ health awareness regarding the prevention and the management of the disease. The current study aimed to evaluate the impact of clinical pharmacist education, counseling and follow up on the management of NAFLD with MetS. Methods: This study recruited 102 patients with NAFLD and MetS. Participants received regular health education sessions concerning the disease and recommended lifestyle (diet and exercise) and were followed by clinical pharmacist for 6 months. At the end of the study, participants were divided into two groups; those compliant with education and follow up session (compliant group; n=61); and those attending > 60% but not completing whole sessions (non-compliant group; n=41). Anthropometric measures, liver function, lipid profile, homeostasis model of assessment-insulin resistance (HOMA-IR), biochemical NAFLD score, radiological liver steatosis and fibrosis grade changes were recorded at baseline and endpoint. Results: The compliant group showed a more significant decrease in weight (p=0.003), low density lipoprotein (LDL) (p=0.009), and very LDL (p<0.001), and a more significant increase in high density lipoprotein (HDL) (p=0.010) compared with the non-compliant group. Moreover, the compliant group showed a statistically significant higher number of patients achieving normalization of total cholesterol (p=0.002), HDL (p=0.004), waist circumference (p=0.004), improvement of body mass index category (p=0.008), liver steatosis grade (p=0.009), liver fibrosis grade (p<0.001), and absence of dyspeptic symptoms (p=0.0010) and hepatomegaly (p= 0.027) compared with the non-compliant group. Fasting blood glucose (p=0.209), fasting insulin (p=0.179), and HOMA-IR score (p=0.193) showed non-significant difference between both groups at endpoint. Conclusion: The educational intervention of a clinical pharmacist showed significant positive impact on ameliorating metabolic syndrome parameters and achieving desired NAFLD therapeutic outcomes.", "keywords": [ "NAFLD", "clinical pharmacist", "patient education", "life style recommendations", "metabolic comorbidities" ], "content": "Background\n\nThe logistic and economic pressure on healthcare systems worldwide have encouraged the incorporation of pharmacists in disease state management services. This is mostly attributed to the fact that pharmacists are one of the most accessible and knowledgeable health care professionals when concerning disease and medication characteristics. Thus they can ensure optimization of acute and chronic patient care.1 The pharmacist has a crucial role in managing many chronic conditions, including heart failure, asthma, chronic obstructive pulmonary disease, diabetes mellitus (DM), hypertension, dyslipidemia, overweight and obesity. Incorporating a pharmacist in the care of such diseases has led to the achievement of target therapeutic goals, prevention and management of drug related problems, and reduction of medication costs.2–9\n\nNon-alcoholic fatty liver disease (NAFLD) includes two pathologically distinct conditions; non-alcoholic fatty liver (NAFL), characterized by simple hepatic steatosis and absence of hepatocyte injury, and non-alcoholic steatohepatitis (NASH), associated with hepatocellular injury with or without fibrosis.10 NAFLD is a health condition with significant morbidity, mortality, and economic burden to the healthcare system.11 In these patients, multiple metabolic comorbidities often exist, such as hypertension, type 2 DM, hyperlipidemia, and overweight or obesity. Complications of NAFLD include liver cell failure, hepatocellular carcinoma and the need for liver transplantation.12 Thus, the goal is not only to treat the liver-specific disease but to manage comorbidities and prevent complications.13 The only FDA-approved anti-NAFLD measure is lifestyle recommendations, including dietary and exercise advice. Thus effective patient education and close follow up is needed. Pharmacists can play a significant role in supporting NAFLD patient education to achieve target anthropometric measures and improve hepatic fibrosis and steatosis.14\n\nPharmacists have the basic knowledge and clinical skills, including prioritization and individualization of therapeutic goals and professional communication abilities, necessary to perform such roles. Unfortunately, clinical pharmacists are struggle to implement their roles within a multidisciplinary team providing patient care in developing countries.15 To our knowledge, studies investigating the impact of pharmacists’ roles in the management of NAFLD are absent worldwide and most of the evidence evaluating the impact of health professional in managing metabolic morbidities focus on physicians and nurses.16 Moreover, evidence supporting pharmacist involvement in NAFLD or metabolic morbidities prevention or management are completely absent in Egyptian population.17 We previously reported the beneficial outcome of adjuvant phosphatidylcholine in Egyptians with NAFLD plus the cornerstone management modality of life style and exercise education conducted by a clinical pharmacist to all study participants.18 As an extension of the previous work, the aim of the current study was to evaluate the impact of a clinical pharmacist education, counseling and follow up in the management of Egyptian NAFLD patients with metabolic co-morbidities.\n\n\nMethods\n\nThis non-randomized interventional study was conducted in the out-patient clinics of the tropical medicine departments at Ain Shams University Hospitals and Ain Shams Specialized Hospital in Cairo, Egypt, between January 2016 and January 2019.\n\nPatients older than 18 year with a diagnosis of NAFLD (including NAFL and NASH) according to the EASL-EASD-EASO Clinical Practice Guidelines19 were included in the study. Recruitment was done through tropical medicine physician appointments for ultrasonography. Patients were excluded from the study if they have an evidence of other types of liver disease (alcoholic, autoimmune, carcinoma), have end stage liver disease, were pregnant or lactating, and had used a lipid lowering or weight loss medication in the last 6 months.\n\nDue to the reporting of multiple primary outcomes a number of least 100 patients for both arms together were considered sufficient to achieve an alpha error of 5% and a beta error of 1%. Patients who consented for any of investigations aim were included in statistical analysis to enhance the significance of results.\n\nSince the approval was released on the basis of supporting lifestyle modifications as counseled and followed up a clinical pharmacist to all study participants,18 grouping for statistical analysis was done at the end of the study duration based on patients compliance to scheduled sessions and resulted in the existence of 61 and 41 participants in compliant and noncompliant group respectively.\n\nThe study protocol was approved by the institutional review board of Ain Shams University according to the declaration of Helsinki. The study was registered in ClinicalTrials.gov under the identifier NCT04411862. Participants were only recruited after they returned a signed informed consent form.\n\nThe interventing clinical pharmacist (the corresponding author) interviewed all eligible participants (n=102) to collect contact details (for follow up), histories, including medical, medication, family, and social histories. All eligible participants were then provided with regular (one every two weeks for six months) health education, counseling and follow up sessions as individualized appointments with the intervening clinical pharmacist in a private consultation room inside out-patient clinics area. The education sessions included an explanation of what NAFLD is and that it is reversible with lifestyle change, the expected complications of inappropriately managed disease and the recommended life-style modifications.37 General advice included aerobic exercise (walking for 30 minutes daily, or > 3 km/day three times weekly) and restricting caloric intake to less than 30 kcal/kg/day while maintaining a balanced diet that included low levels of saturated and trans-fats to achieve gradual weight loss. The target weight loss was a 10% loss within 6 months to avoid rapid weight loss (> 1.6 kg/week) that could increase the progression of NAFLD. Self-regulation was encouraged through participants counselling about regular body weighing, using a smartphone app or internet websites, or even a dairy for counting daily calories, and use of pedometers or body activity trackers. Additionally, a WhatsApp group including all participants and the intervening clinical pharmacist was established. Replies to participant’s inquiries were provided through this group as well as through the interventing clinical pharmacist’s email (provided to patients) for providing individualized patient counseling. The clinical pharmacist also provided participants with a written scheduled appointments for sessions, and educative brochures describing the nature and complications of NAFLD and comorbidities such as diabetes, obesity, and metabolic syndrome, as well as individualized monthly dietary plans for all recruited patients. Participants were encouraged to provide a food and exercise log.20\n\nParticipant recruitment, grouping, intervention and follow up are summarized in the CONSORT flow chart (Figure 1).\n\nBMI: Body mass index; FBG: Fasting Blood Glucose; FBI: Fasting Blood Insulin; HOMA-IR: Homeostasis Model of Assessment-Insulin Resistance; NAFLD: Non Alcoholic Fatty Liver Disease.\n\nParticipants were grouped according to their compliance to the follow up and education sessions: compliant group, those who attended all 12 sessions (n=61); and non-compliant group, those who attended more than 8 sessions but did not complete all 12 sessions (n=41).21 No participants attended less than 8 sessions.\n\nStudy outcomes were measured at baseline and endpoint in both groups. The primary endpoint for NAFLD was the change of NAFLD radiological parameters measured by abdominal transient elastography, using FibroScan® Expert 630 (Echosens, Paris, France) to measure liver fibrosis and steatosis. Liver fibrosis grades included: F0 referring to no fibrosis, F1 referring to portal fibrosis without septa, F2 referring to portal fibrosis with few septa, F3 referring to numerous septa without cirrhosis, and F4 referring to cirrhosis.22 Liver steatosis grades included: S0 referring to <5% steatosis, S1 referring to a 5-33% steatosis, S2 referring to a 33-66% steatosis, and S3 referring to >66% steatosis.23\n\nThe primary endpoint for metabolic co-morbidities was the number of patients with MetS defined by the presence of any three out of the following five features: waist circumference in the horizontal plane midway between the lowest ribs and the iliac crest24 ≥102 cm in men or ≥88 cm in women; triglycerides (TG) ≥150 mg/dL; high density lipoprotein-cholesterol (HDL) <40 mg/dL in men or <50 mg/dL in women; blood systolic pressure ≥130 or diastolic >85 mm Hg measured according to the standard technique using validated cuff-based automated devices in a properly configured setting25 or on antihypertensive drug in patients with a history of hypertension; and fasting blood glucose (FBG) >100 mg/dL or on drug treatment for elevated glucose (including diabetics).26 TG, HDL, and FBG were measured by enzymatic colorimetric method with the Olympus Corp. AU 600 autoanalyser using reagents from Olympus Corp. (Hamburg, Germany).\n\nSecondary outcome measurements included anthropometric changes to body mass index (BMI, calculated as weight divided by squared height; weight and height were measured with a Health-ometer professional scale), waist and hip circumference, biochemical markers of NAFLD, and comorbidities such as liver functions (determined by colorimetric assay using kits obtained from Biomerieux Vitek, Inc Missouri USA), lipid profile, and fasting insulin (measured by chemiluminescent immunoassay method; Immulite 1000 System, DPC, Los Angeles, CA; 90045-5597) and blood glucose. Additionally, NAFLD fibrosis score (a combined biochemical and clinical score calculated using platelet count, serum aminotransferases and albumin, age, BMI, and diabetic status, calculated using NAFLD fibrosis score calculator) that estimate the probability of liver fibrosis,23 and homeostasis model assessment—insulin resistance (HOMA-IR; a biochemical score calculated using serum fasting blood glucose and insulin) score that assess insulin resistance in non- diabetic patients27 were estimated for each participant using the HOMA2 Calculator (University of Oxford, 2019). NAFLD fibrosis score categories includes: low cutoffs (<-1.455), predicting a fibrosis level of F0-F2; indeterminate cutoffs (-1.455 to 0.675); and high cutoffs (>0.675) predicting a fibrosis level of F3-F4.13 A HOMA-IR >3 indicates the presence of insulin resistance.27\n\nRecorded data were analyzed using the SPSS, version 20.0 (SPSS Inc., Chicago, Illinois, USA, RRID:SCR_002865). Data were explored for normality using the Kolmogorov-Smirnov test.28 Quantitative data were expressed as mean± standard deviation if parametric and as median and interquartile ranges if non-parametric. Qualitative data were expressed as frequency and percentage. Independent-samples t-tests were used to compare two samples of quantitative parametric means. Post-hoc comparisons were conducted using the Bonferroni correction method. The chi-square test was used to compare qualitative parameters. The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the p-value was considered significant if <0.05 and highly significant if <0.001.\n\n\nResults\n\nThe mean levels and comparisons of selected baseline demographics, weight and metabolic co-morbidities of studied groups are shown in Table 1. The study cohort (n=102) included 86 women and 16 men. There was no statistically significant difference between both groups regarding any of the compared demographics.\n\n* p-value >0.05 is non-significant.\n\n** Use essential phospholipids.\n\nThe compliant group showed a statistically significant higher number of patients with normalized waist circumference (p=0.004, Figure 238) and an improved BMI category, as shown in Table 2. These results were accompanied by a decrease in participant weight in kg in both groups but this was more significant in the compliant group (p=0.003) and confirmed with the percent change of 11.66%±5.77 in compliant versus 8.39%±6.05 in non-compliant group; p=0.007).\n\nNAFLD; non-alcoholic fatty liver disease.\n\nStatistically significant difference p≤0.05 as compared to non-compliant.\n\n p-value >0.05 is non-significant.\n\n** Normal weight (18.5 – 24.9 kg/m2), overweight (25-29.9 kg/m2), obese I (>30 kg/m2), obese II (35-39.9 kg/m2), and obese III (>40 kg/m2).\n\nIn the compliant group, at the end of the study, there were significantly lower serum levels of low-density lipoprotein (LDL in mg/dl calculated by Friedewald’s formula; p=0.009), and very low-density lipoproteins (VLDL in mg/dl; p≤0.001). LDL decreased by a mean percent change of 19.93% from baseline in the compliant group compared to 11.41% in the non-compliant group, with a statistically significant difference between groups (p=0.002). The number of patients that reached normal LDL levels (<130 mg/dl) was not significantly higher in the compliant group (p=0.136; Figure 338). TG decreased by a significantly higher mean percent change of 16.48% from baseline in the compliant group compared to 6.24% in the non-compliant group, with a statistically significant difference between groups (p<0.001).HDL (mg/dl) increased by 18.57%±15.18 in the compliant group and 16.40%±19.01 in the non-compliant group but was significantly higher (p=0.010) in compliant group at the endpoint. At the end of the study, significantly higher percentage of compliant participants attained a normalized HDL (>50; p=0.004), and normalized serum total cholesterol levels (<200 mg/dl; p=0.002; Figure 3). At the end of the study, there were no significant differences between studied groups regarding FBG (mg/dl; p=0.209), fasting insulin (FI; μU/ml; p=0.179), HOMA score (p=0.193), serum albumin (P=0.052), AST (P=0.115) and ALT (p=0.405). Also the percentage change from baseline was non-significant for these variables in the compliant group (p=0.073 for FBG, p=0.221 for FI, and p=0.089 for HOMA score).\n\nALT; Alanine Aminotransferase; AST: Aspartate Aminotransferase, FBG: Fasting Blood Glucose; FBI: Fasting Blood Insulin; HOMA-IR: Homeostasis Model of Assessment-Insulin Resistance; HDL-C: High density Lipoproteins-cholesterol; LDL-C: Low Density Lipoproteins-cholesterol; TC: Total Cholesterol; TG: Triglycerides,\n\nNormal ranges: AST= 10-42 U/L; ALT (N=10-45U/L); TC<200 mg/dl; TG <150mg/dl; LDL <130 mg/dl and HDL; >50 mg/dl.\n\n*Statistically significant difference p≤0.05 as compared to non-compliant.\n\nAt the endpoint, as seen in Figure 2,38 the compliant group showed a statistically significant higher number of participants with absent dyspeptic symptoms (p<0.0010), hepatomegaly (p= 0.027) and also with improved NAFLD fibrosis score (p=0.0047), radiological steatosis (p=0.009) and fibrosis (p<0.001) grades.\n\n\nDiscussion\n\nThere is no doubt that disease state management by a multi-specialty-health care team has a multitude of advantages, including reducing the pressure on healthcare systems and attainment of improved health care outcomes both to patients and health care institutions. NAFLD is one of the diseases that can benefit the most of being handled by a multidisciplinary approach and this is due to its multiple comorbidity nature.29 Pharmacists are considered one of the most trustworthy and accessible health care professionals, and are ideally situated to provide patient counseling regarding disease state, life style changes and medication.1,30 Pharmacists have a crucial role in enhancing patient health and medication literacy, thus ensuring patient concordance.17\n\nThe AASLD recommendation for management of NAFLD prioritizes weight loss via dietary modification alone or with exercise.31 The impact of managing both NAFLD and metabolic co-morbidities chiefly include improvement of anthropometric and metabolic parameters Previous studies have reported a positive impact of incorporation of a pharmacist in patient care, in terms of achieving desired goals, reverting to non-metabolic syndrome status, and improved lifestyle modification.32–34\n\nIn the current study, the more significant reduction in participant anthropometric measures in the compliant group, including weight reduction (p=0.003), shifting to an improved BMI category (p=0.008) and waist circumference normalization (p=0.004), was in accordance with the results of several pharmacist-led interventions. A previous systematic review has evaluated the effectiveness of community pharmacist’ weight management interventions, including dietary and physical activity recommendations and follow up in obese patients with obesity related co-morbidities including diabetes, hypertension, depression and gastroesophageal reflux disease. The review reported a statistically significant weight loss at 6 months in three studies.35 The three studies showed an average weight loss of 5.6 kg, 5.1 kg and 5-kg, all much less compared to the average weight loss of 10 kg in the compliant group and 8.5 kg in the non-compliant group in the current study.\n\nSimilarly, in a study that enrolled obese patients with comorbidities including DM, hypertension, and/or dyslipidemia, a six month pharmacist led weight loss intervention resulted in a statistically significant reduction in weight (mean loss of 5 kg representing 4.5% from baseline mean, p<0.001), BMI (p<0.001) and waist circumference (p=0.002).36 This is again less than the findings of the current study, which showed a mean percent weight loss of 11.66%±5.77 in the compliant group and 8.39%±6.05 in the non-compliant group, with a significance of p=0.007.\n\nIn a study conducted by Hammad et al (2011) in Jordan, to compare the effect of a pharmacist-physician collaborative practice versus usual care in 199 patients with metabolic syndrome, 39.1% of patients receiving pharmacist recommendations and counseling versus 24.7% of usual care patients were successfully shifted from a status of metabolic syndrome to no metabolic syndrome (p=0.032).32 Similarly, the current study showed a reduction in the number of patients with metabolic syndrome at the end of the study, although this was non-significant (p=0.37).\n\nConcerning the laboratory outcomes there were also some studies that agree with the findings of the current study. The non-controlled study conducted by Cording et al on 115 patients with dyslipidemia receiving treatment with a statin (57%), fibrates (17%) and those receiving no lipid-lowering medications (17%).3 After 12 months, LDL level decreased by 20%, HDL cholesterol increased by 11%, and triglycerides decreased by 19% relative to baseline. Overall, LDL goals were reached in 77% of the patients.3 Similarly, in the current study, a significantly higher number of compliant participants converted to normal serum total cholesterol levels compared to non-compliant participants (p=0.002) at the end of the study. The results of the current study are also in accordance with those obtained by Hammad et al, where the mean TG serum level declined by 30.9 mg/dl in the intervention group and by 14.5 mg/dl in the usual care group (p=0.029).32\n\nThe positive radiological outcomes of NAFLD obtained in the current study by the pharmacist led counseling and education can’t be compared to similarly conducted studies, as to our knowledge there are none. The higher rate of therapeutic goal achievement in the current study and similarly conducted studies could be attributed to the quality time and individualized attention spent in group and individual patient education, counseling and follow-up that ensured better patient understanding of the disease process, possible complications of inappropriately managed disease and necessary lifestyle changes to manage the disease.\n\nThe cost effectiveness of providing this service was not evaluated in the current study. Highlighting the potential long-term healthcare cost reduction secondary to clinical pharmacist implementation into multidisciplinary health team in the face of the expenses of recruiting clinical pharmacists, as well as the identifying the acceptance rate of pharmacist role by non-pharmacist professionals, can reinforce clinical pharmacist collaboration. Future larger scale research is warranted to evaluate the impact of clinical pharmacist collaboration in the management of NAFLD and metabolic morbidities.\n\n\nConclusions\n\nThe current study has provided significant evidence of the benefit of incorporating a clinical pharmacist in NAFLD patient counseling, education and follow up. This significantly facilitates reaching desired therapeutic goals of NAFLD and metabolic co-morbidities. Thus, the current study indicates that clinical pharmacists could be viable health care providers for such a patient population especially in the face of shortage of primary care provider time in developing countries.\n\n\nData availability\n\nFigshare: Dr Nehal DATA.xlsx, https://doi.org/10.6084/m9.figshare.17104499.37\n\nThis project contains the following underlying data:\n\n• Dr Nehal DATA.xlsx (Raw DATA for compliant and noncompliant group)\n\nFigshare: Excel sheet of figures https://doi.org/10.6084/m9.figshare.18953414.38\n\nThis project contains the following extended data:\n\n• excel of fig 2.xlsx\n\n• excel of fig 3.xlsx\n\nFigshare: Health education material and diet schedule for NAFLD https://doi.org/10.6084/m9.figshare.18866309.20\n\nThis project contains the following extended data:\n\n• Health Education Material.docx (Health education material in Arabic)\n\nFigshare: CONSORT checklist, https://doi.org/10.6084/m9.figshare.18866306\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nWritten informed consent was obtained from all the patients to participate in clinical research and for publication of the patients’ clinical details. This research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.", "appendix": "References\n\nMaulavizada H, Emmerton L, Hattingh HL: Can a pharmacy intervention improve the metabolic risks of mental health patients? Evaluation of a novel collaborative service. BMC Health Serv. Res. 2016; 16(1): 146. PubMed Abstract \| Publisher Full Text\n\nRamser KL, et al.: Physician-Pharmacist Collaboration in the Management of Patients With Diabetes Resistant to Usual Care. Diabetes Spectr. 2008; 21(3): 209–214. Publisher Full Text\n\nCording MA, et al.: Development of a pharmacist-managed lipid clinic. Ann. Pharmacother. 2002; 36(5): 892–904. PubMed Abstract \| Publisher Full Text\n\nCarter BL, et al.: Physician and pharmacist collaboration to improve blood pressure control. Arch. Intern. Med. 2009; 169(21): 1996–2002. 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Figure.2022." }	[ { "id": "125472", "date": "11 Mar 2022", "name": "Giovanni Tarantino", "expertise": [ "Reviewer Expertise NAFLD", "Metabolic Syndrome", "Atherosclerosis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 investigated the impact of clinical pharmacist-led health education on the disease course of non-alcoholic fatty liver disease patients. The topic is interesting, and the manuscript is well written. The introduction covered the main ideas investigated in the study. All data of surface roughness are available. The discussion explained the results thoroughly.\nHowever, some minor comments are suggested for the authors to consider.\nAuthors should comment on the fact that HOMA did not show any change between the two groups, even though several other parameters changed significantly, at the light that insulin resistance is the driver of NAFLD and for some other authors is the consequence as clearly presented in Gastaldelli (2017).1\nIn the discussion, give a brief account of the limitations of your study. This paragraph should be expanded pointing on the lack of determination of some inflammatory indices such as cytokines, about which there are a lot of pieces of research, specifically interleukin-17, linking NAFLD to the most frequent co-morbidity that is atherosclerosis as evident.23\nThe authors also did not mention sample size/power calculation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "143241", "date": "27 Jul 2022", "name": "Doaa El Amrousy", "expertise": [ "Reviewer Expertise pediatric gastroenterology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\ni reviewed with interest the study titled \"The impact of clinical pharmacist-led health education on the disease course of non-alcoholic fatty liver disease patients: an interventional study\" which aimed to evaluate the impact of clinical pharmacist education, counseling and follow up on the management of 102 p-atients NAFLD with MetS. the study is well designed, well written and the methodology was sound. but i have few comments:\nAbbreviations should be defined the first time they are mentioned e.g EASL.....\n\nStudy design: it appears like a clinical trial to evaluate the effect of pharmacist health education on patients with NAFLD. Did you register this trial?\n\nSample size details is needed e.g. on which outcome you estimate the sample size and what is the effect size? Program used?\n\nSecond paragraph under the title \"sample size\" is not related to this title at all. It is actually related to study intervention and follow up.\n\nUnder \"at the end of the study\" title: you mentioned the number of patients who were compliant and who were not. this is result and is not appropriate to be mentioned here.\n\n\"Outcomes\": most of the details under this study is related to the study measurements and should not be mentioned here e.g radiological details. just mention the outcomes without mention the mesaurements details which should be moved under the title \" study measurements\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "143239", "date": "27 Jul 2022", "name": "Bernhard Langer", "expertise": [ "Reviewer Expertise Pharmacy Practice", "Health Policy", "Health Care Financing", "Quality Improvement" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany thanks to F1000Research for letting me review this interesting article. I enjoyed reading the manuscript. However, I have many comments for improvement. Pages in this text refer to the pdf file of the manuscript.\nTRIAL REGISTRATION:\nPage 3: why did you not register the trial before the beginning of the data collection?\nTITLE:\nAdequate.\nABSTRACT:\nIn general, adequate.\nPage 2: \"…dyspeptic symptoms (p<0.0010)…” You mean p<0.001, right?\nBACKGROUND:\nIn general, very well written with a very clear argumentation and expression.\nPage 3: you mention an FDA-approved measure, but are there any measures/guidelines in Egypt? If so, what is the content?\nPage 3: \"...health professional...\" 's' is missing, right?\nMETHODS:\nPage 3: you should add the fact that the study is not randomized to the limitations. Why did you not opt for a randomized study design? Is the reason perhaps that the group division should then have been done at the beginning of the study, but compliance cannot be guaranteed? Perhaps you could also discuss this or other reasons in the limitations.\nPage 3 and 4: the study described under Clinicaltrials.gov NCT04411862 is not the study reported in this paper, right? But why did you mention the registration in Clinicaltrials.gov? In your previous work (same trial?) published in reference 18, you reported that the study was conducted between January 2015 and January 2019, in your manuscript this timeline is between January 2016 and January 2019. Please revise accordingly.\nThe relation between the two studies (study mentioned in reference 18 and this study) is unclear. Please explain it more detailed.\nPage 3: please describe the recruitment process in a little more detail.\nPage 3: “Due to the reporting of multiple primary outcomes a number of least 100 patients for both arms together were considered sufficient to achieve an alpha error of 5% and a beta error of 1%.” Could you please cite an appropriate reference for this statement? Why didn't you perform a formal sample size calculation (formula or G*Power software)?\nPage 4: The question is how sustainable the intervention is. For this purpose, it would have made sense to conduct a further follow-up after, say, another three or six months. Or do the authors assume or suggest that the intervention should be \"permanent\"? In this case, however, the question of the costs of the intervention or the cost-benefit ratio arises all the more. Perhaps the authors should add these aspects to the limitations.\nPage 4: MetS: Please write out and abbreviation in brackets, as mentioned for the first time in the main text.\nPage 4: “…hip circumference…” This outcome is not reported in the RESULTS section. Please revise accordingly.\nPage 5: I believe that a consistent specification of effect sizes (Cramer's V for categorical data and, for example, Cohen's d for continuous data) could improve your analysis. Please also provide an appropriate interpretation according to Cohen (1992).1 See also: https://www.socscistatistics.com/effectsize/default3.aspx.\nPage 5: “…for normality using the Kolmogorov-Smirnov test.” Yes, but you did not specify whether the data is normally distributed or not and what conclusion to draw from it. Please revise accordingly.\nPage 5: “Quantitative…” Wrong expression: you mean \"Continuous\", right?\nPage 5: “…and as median and interquartile ranges if non-parametric.” Yes, but in the RESULTS, you always report mean and SD. Thus, why did you mention median and IQR in this section?\nPage 5:” Qualitative…” Wrong expression, you mean \"Categorial\", right?\nPage 5: “Independent-samples t-tests were used to compare two samples of quantitative parametric means.” Better: “Parametric independent t-tests were used to determine whether there is a statistically significant difference between the means in two unrelated groups.”\nPage 5: “Post-hoc comparisons were conducted using the Bonferroni correction method.” Perhaps you could explain in a bit more detail why you made an adjustment for multiple testing (perhaps multiple primary endpoints?). You should also provide an appropriate reference, for example Bender and Lange (2001).2 Also, you have not reported Bonferroni corrections for the P values in your RESULTS section. Please revise accordingly.\nPage 5: \"…qualitative…“ Wrong expression, you mean \"categorial\", right? Please revise accordingly.\nPage 5: “The chi-square test was used to compare qualitative parameters.” Better: “Chi-Square Tests were used to determine whether there is an association between categorical variables.” Please revise accordingly.\nRESULTS:\nPage 6: In general, reporting of results can be improved because it is sometimes confusing (e.g., text and Figure 3).\nPage 6, table 1: Is it possible to compare the compliant and non-compliant group with respect to other baseline variables? Please revise accordingly.\nPage 6, table 1: There is no space between some numbers. Please revise accordingly.\nPage 6, figure 2: Please always show three decimal places. Please revise accordingly.\nPage 6, figure 2: In the figure you use the wording “Fibrosis stage”, in the text you use the wording “fibrosis grade”. Please revise accordingly.\nPage 6, figure 2: “Dyspeptic syndrome presence”. This outcome variable was not mentioned in the METHODS. Please revise accordingly.\nPage 6, figure 2: “Heptomegy presence”. Are you sure that this is the right wording? Also, this outcome variable was not mentioned in the METHODS. Please revise accordingly.\nPage 7, figure 3: On the x-axis, FI is reported, but in the legend, you reported FBI. Please revise accordingly.\nPage 7, legend of figure 3: Reporting of normal ranges for VLDL is missing. Please revise accordingly.\nPage 8: “…(p<0.001).HDL…” There is no space between the two sentences. Please revise accordingly.\nPage 8: “…fasting insulin (FI;…” FI or FBI? Please revise accordingly.\nPage 8: \"…dyspeptic symptoms (p<0.0010)…” You mean p<0.001, right?\nPage 8: “…improved NAFLD fibrosis score (p=0.0047)…” But in Figure 2 you reported a P-value of 0.049. Please revise accordingly.\nPage 8: “…fibrosis (p<0.001) grades.” But in Figure 2, you reported a P-value of 0.66, right? Please revise accordingly.\nDISCUSSION:\nGenerally, well written. Avoid reporting detailed results already listed in the RESULTS section. Please revise accordingly.\nPage 8: “The impact of managing both NAFLD and metabolic co-morbidities chiefly include improvement of anthropometric and metabolic parameters”. Full stop is missing. Please revise accordingly.\nPage 8: “…all much less compared to the average weight loss of 10 kg in the compliant group and 8.5 kg in the non-compliant group in the current study.” Avoid reporting results in the discussion that were NOT reported in the RESULTS section. Please revise accordingly.\nCONCLUSIONS:\nAdequate.\nREFERENCES:\nReference 11 and 12: why is there no PubMed link, because this journal is indexed by PubMed since 1981?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-234
https://f1000research.com/articles/11-233/v1	24 Feb 22	{ "type": "Software Tool Article", "title": "miND (miRNA NGS Discovery pipeline): a small RNA-seq analysis pipeline and report generator for microRNA biomarker discovery studies", "authors": [ "Andreas Diendorfer", "Kseniya Khamina", "Marianne Pultar", "Matthias Hackl", "Andreas Diendorfer", "Kseniya Khamina", "Marianne Pultar" ], "abstract": "In contrast to traditional methods like real-time polymerase chain reaction, next-generation sequencing (NGS), and especially small RNA-seq, enables the untargeted investigation of the whole small RNAome, including microRNAs (miRNAs) but also a multitude of other RNA species. With the promising application of small RNAs as biofluid-based biomarkers, small RNA-seq is the method of choice for an initial discovery study. However, the presentation of specific quality aspects of small RNA-seq data varies significantly between laboratories and is lacking a common (minimal) standard. The miRNA NGS Discovery pipeline (miND) aims to bridge the gap between wet lab scientist and bioinformatics with an easy to setup configuration sheet and an automatically generated comprehensive report that contains all essential qualitative and quantitative results that should be reported. Besides the standard steps like preprocessing, mapping, visualization, and quantification of reads, the pipeline also incorporates differential expression analysis when given the appropriate information regarding sample groups. Although miND has a focus on miRNAs, other RNA species like tRNAs, piRNA, snRNA, or snoRNA are included and mapping statistics are available for further analysis. miND has been developed and tested on a multitude of data sets with various RNA sources (tissue, plasma, extracellular vesicles, urine, etc.) and different species. miND is a Snakemake based pipeline and thus incorporates all advantages using a flexible workflow management system. Reference databases are downloaded, prepared and built with an included (but separate) workflow and thus can easily be updated to the most recent version but also stored for reproducibility. In conclusion, the miND pipeline aims to streamline the bioinformatics processing of small RNA-seq data by standardizing the processing from raw data to a final, comprehensive and reproducible report.", "keywords": [ "microRNA", "Next-Generation Sequencing", "differential expression", "smallRNA sequencing", "biomarkers", "spike-in", "discovery study" ], "content": "Introduction\n\nSmall RNA-seq has been a well-established tool for the quantification of short RNA molecules like microRNAs (miRNAs) in various biofluids (Murillo et al., 2019). Those short RNA molecules (17 to 25nt) play an important role in the cellular regulation of gene expression by interacting with specific complementary sites in targeted messenger RNAs (mRNAs). mRNAs that contain these target sites are then either down- or (rarely) up-regulated, resulting in a regulatory effect on the downstream translation of the mRNA (O’Brien et al., 2018). In this context, miRNAs are part of a complex regulatory network where their expression does not only affect other mRNAs, but also the expression of miRNAs themselves is highly controlled (Lee & Ambros, 2001). Thus, the levels of miRNAs can be indicators of a cell’s regulatory state and correlate with an organism’s health status. For example the liver specific miR-122-5p was shown to be a suitable marker for liver injury when measured in serum or plasma (Llewellyn et al., 2021) and as part of a miRNA expression signature can even be used to predict recovery after liver resection (Starlinger et al., 2019).\n\nThis makes them interesting targets as biomarkers in liquid biopsy (Larrea et al., 2016). But the search for miRNAs or miRNA signatures that are suitable as biomarkers requires a very specialized approach regarding computational biology. As next-generation sequencing (NGS) is often used in the discovery phase of studies (de Ronde et al., 2018), a standardized and specialized analysis pipeline is highly important. Existing tools and pipelines for small RNA NGS analysis (like miRDeep2, miRExpress, miRNAkey, and sRNAbench) mostly focus on single steps, like the quantification of miRNAs or differential expression profiling, but either not provide additional analysis such as unsupervised analysis methods and data quality checks, or only in hard to interpret and inaccessible ways (Friedländer et al., 2012; Wang et al., 2009; Ronen et al., 2010; Aparicio-Puerta et al., 2019).\n\nWith the need for a standardized report that contains all relevant data and an initial statistical analysis, we developed a small RNA-seq data processing pipeline that not only provides one centralized report with all relevant information, but also bridges the gap between biologists and bioinformaticians with very easy to prepare data submission files as input and a detailed and well documented and interactive report as output.\n\nIn this study, we developed a robust and portable analysis pipeline for NGS data with a focus on biomarkers in discovery studies. With this in mind, we targeted the following goals: (1) standardized data inputs, (2) reproducible analysis, and (3) ease of use for both bioinformaticians and study statisticians (including publication ready figures and a clear and intuitive representation of results).\n\nThe miND pipeline can be used on many operating systems and in various setups with the only requirement of being able to run Snakemake workflows (Köster & Rahmann, 2012). Wrapper scripts for startup of the pipeline on Linux based systems are provided which can be adapted for the use on different platforms.\n\n\nMethods\n\nThe pipeline is based on Snakemake (Köster & Rahmann, 2012), a scalable bioinformatics workflow engine which incorporates many features needed for reproducible computational analysis (Mölder et al., 2021). This includes handling the installation and provisioning of software tools via conda (“Anaconda Software Distribution,” 2020) and bioconda (Grüning et al., 2018) and overall the orchestration of individual steps of the pipeline to optimize usage of limited resources like central processing unit (CPU) and memory. Configuration files in yml format are used and contain settings for multithreading to adapt the pipeline for various computing platforms (Diendorfer et al., 2022).\n\n\nUse case\n\nAn example protocol demonstrating the analysis of a public data set is available at protocols.io under the name miND pipeline AWS EC2 installation and setup V.2 and can be reproduced not only as a guide for following data analysis, but also to setup the pipeline and data repository. The protocol describes the setup in an Amazon Web Services EC2 (Amazon Web Services, Inc, 2015) instance but has also been developed and tested on other platforms and systems. Only operating system specific parts would have to be adapted (e.g., installation of tools like git or wget would be done via apt on Debian based Linux distributions). For scientists interested in running the miND pipeline themselves, it is highly recommended to follow the provided protocol with the example data before running analysis on their own data sets.\n\nThe generated miND report for this example data set is available on GitHub.\n\nThe miND pipeline was developed and tested on Debian Linux (v11.2) running Snakemake (v6.0.5) and conda (v.4.10.3). The hardware requirements depend on the size of analyzed datasets, but in general it is recommended to provide at least 4 CPU cores and 8GB of memory. The pipeline will scale according to the available resources.\n\nThe pipeline requires data from three reference data sets: (1) host genomes from ENSEMBL (Zerbino et al., 2018), (2) RNA sequences from RNAcentral (Sweeney et al., 2019), and (3) miRNA mature and precursor sequences from miRbase (Griffiths-Jones, 2004).\n\nIn order to download and prepare these datasets in the formats and structures required, miND provides separate workflows to build the data repository. These workflows can be executed with a shell script that will read configurations for each data source and then download, format and build the reference databases based on Snakemake workflows.\n\nThe data repository only has to be built once and will then provide the data needed for all future miND analysis runs. In case of updates of reference data sets, the repository can be rebuilt or extended by adding sources to the configuration files and running the build script again.\n\nThe miND pipeline requires two types of data for each experiment: raw NGS data and a meta data file with additional sample information. Raw data can be supplied either in fastq, fastq.gz or BAM (without alignments) files. The given format will be detected based on the file extensions.\n\nExperimental meta data and details about the samples is provided in a XLS file containing three sheets: (1) Project details sheet, with general information and data of the project. This includes project title and comments but also settings relevant for the processing of the data like the sample species, adapter sequences, and cutoff levels for significance and quality filtering. (2) Sample group matrix sheet, which lists all samples that are part of this experiment and links them to additional group information. Up to five grouping variables can be set with unlimited levels each. The last sheet contains the (3) Contrast selection and allows the selection of groups and group-combinations based on the data provided in the sample group matrix sheet. The contrasts selected here will be used for the differential expression analysis.\n\nThe overall flow of data through the pipeline is shown in Figure 1. This flow diagram outlines the most important steps of data processing in the miND pipeline, especially the quality control steps with FastQC (Andrews, 2010) and multiQC (Ewels et al., 2016), followed by hierarchical mapping using bowtie1 (Langmead et al., 2009) and miRDeep2 (Friedländer et al., 2012), where either mapped or unmapped reads are further processed by the next step. The final “R scripts processing” step includes multiple scripts that preprocess and analyze that data (including mapping statistics, unsupervised analysis methods and differential expression analysis) to then generate an interactive HTML report based on R markdown.\n\nReference data is downloaded and processed by the repository build process (yellow area; top right) and then available for the miND pipeline in the repository/subfolder. Raw next-generations sequencing (NGS) data (blue area) is first adapter and quality trimmed and then handled by quality control (QC) tools and processed through hierarchical mapping steps (green area). These steps produce a set of mapping files that are then ingested and analyzed by R scripts, producing the miND report in the end.\n\nThe hierarchical mapping uses genome datasets from the prepared data repository (generated once before the initial run as described in the “Data repository” subsection) in a first step to filter out reads that to not map to the host organism’s genome (bowtie1, allowing for two mismatches). The genome-mapped reads are further processed by miRDeep2 to accurately quantify miRNAs. To identify further remaining (genome mapping but non-miRNA) reads, bowtie1 is used to first map against the RNAcentral database and then complementary DNA sequences (to assign mRNA reads), both steps allowing for one mismatch. Reads that remain unmapped after these hierarchical clustering are classified as either “unknown genomic” (if they mapped against the host genome) or “unmapped” (in case of reads that did not map against the host genome and were thus filtered in the first mapping step). The generated mapping files are processed by R scripts to prepare mapping statistics for the different RNA species in each sample.\n\nThe mapping process focuses on miRNAs and prioritizes them by using the specialized mapping tool miRDeep2 directly after an initial genome mapping step. It utilizes bowtie1 for mapping of the reads but performs a more sophisticated assignment of miRNA IDs to the reads. This includes detailed information of isomiRs (mature miRNAs with highly similar sequences) that is prepared for further analysis steps.\n\nFor the identification of other RNA species RNAcentral is used. This comprehensive database contains non-coding RNA (ncRNA) sequences from a broad range of species. This step focuses on the classification of reads and uses bowtie1 (allowing for one mismatch) reporting the first (best) hit. This limits the use of the mapping data to the required classification, as reads could map to multiple references which are not reported mainly for performance reasons.\n\nmiND pipeline uses the popular R package EdgeR (Robinson et al., 2009) for differential expression analysis (DEA) with the quasi-likelihood negative binomial generalized log-linear model functions provided by the package.\n\nA central role of NGS data processing and especially in DEA is the filtering of reads with low expression levels. Those reads would otherwise increase the noise level in the data and result in a high rate of false positives in the following DEA. Recommendations on fixed reads per million reads (RPM) based cutoff values (e.g., filtering all reads with less than 10 RPM) do not adequately account for variations in library size and miRNA reads ratio in the library and are thus arbitrary cutoffs. The DEA package DESeq2 (Love et al., 2014) implements an independent filtering method that was adapted in miND to be used also with EdgeR. Assuming that most false-positives are caused by low abundant miRNAs, the algorithm removes quantiles of miRNAs from the low-abundance end and checks if the number of significant miRNAs increases after false-discovery rate (FDR) adjustment. This would be the case if mostly false positives have been removed because FDR adjustment would now be more sensitive and not remove as many true positives, increasing the overall number of significant results. This method works reliably if there are any true positive results. If the result set consist only of false positives, then even after removing the low abundant miRNAs, results would not increase the number of significant results (as there are no true positives to enrich). In this case our implementation of the algorithm has a fallback, where very lowly expressed miRNAs are pre-filtered prior to DEA and FDR adjustment: In a first step, miRNAs that are only expressed at very low levels are filtered, which is defined as RPM values that are lower than 10 divided by the smallest library size in at least half the number of samples of the smaller group. Those miRNAs carry no biological and statistical relevance (Chen et al., 2016) as they have very low read counts in both groups.\n\nAn exemplary relation between a given quantile cut-off and the resulting number of differentially expressed miRNAs after FDR is shown in Figure 2.\n\nEach point represents the number of differentially expressed micro ribonucleic acids (miRNAs) after false discovery rate (FDR) adjustment and done in steps of increasingly stringent quantile-based reads filtering. With more and more low read count miRNAs removed from the differential expression analysis, the number of significant (FDR) differentially expressed (DE) miRNAs increases to the point where more and more true positives get removed, thus decreasing the total amount of DE miRNAs. This is shown in the graph as the maximum of the red line. The optimal quantile cutoff value is then determined by finding this maximum.\n\nFor differential expression the contrasts of interest can be selected in the experiment meta data XLS file (last sheet of the SampleContrastSheet.xlsx). Either groups or combinations of groups can be selected based on the group information provided for the samples. Each selected contrast will be part of the final interactive HTML report. In addition, a blocking factor can be selected if applicable. This blocking factor will be included in the model for the differential expression as additive factor and thus can be used e.g., for a paired experimental design or to account for batch effects.\n\nAlthough DEA is a central point of biomarker discovery studies, other statistical methods are needed to put this analysis into context and ensure valid results. The miND pipeline report contains a series of additional graphs and tables to present the data in a way that is interactive and easy to browse. The main sections (see Figure 3) are (1) introduction, (2) data exploration (including a sample table, reads classification plots, miRNA mapping tables, heatmaps, principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE plots)), (3) differential expression results, and (4) an appendix (references and run information).\n\nThe main sections (1) introduction, (2) data exploration, (3) differential expression, and (4) appendix each contain multiple subsections. The standardized structure of the report allows for the quick assessment and comparison of experiment results. t-distributed stochastic neighbor embedding (t-SNE), micro ribonucleic acids (miRNAs), differentially expressed (DE).\n\nReads classification plots\n\nThe reads classification plots (see Figure 4) present the amounts of reads mapped to different RNA species (miRNAs, tRNAs, piRNA, rRNA, lncRNA, etc.) based on the hierarchical mapping done by the miND pipeline. This is plotted as absolute reads but also as relative ratios (percent) to get a quick impression of the RNA classes that are present in the data set. Especially for samples with low numbers of miRNAs present (e.g. extracellular vesicles) these two graphs give important information about the success of library preparation and sequencing.\n\nEach bar represents an individual sample, while colors of the bar charts give insights in the mapped ribonucleic acid (RNA) species. This representation helps with a quick identification of library prep or sequencing issues if the ratios or total number of reads are not as expected.\n\nThe data on which the reads classification plots are based on is also included in the HTML report and can be either browsed directly in the HTML file or (as all tables and figures) or exported in various data formats (CSV or XLS for tabular data and PNG for graphics) for further analysis or publications.\n\nmiRNA mappings table\n\nThe miRNA mappings table contains read counts for each miRNA that was found in at least one of the samples. The table is available with raw read counts but also as RPM (normalized to the total number of miRNAs mapped in each sample). Group information is included in this table, if provided by the experiment metadata XLS file.\n\nA visualization of the miRNA mapping statistics helps in comparing the number if identified miRNAs in the samples (see Figure 5). For each sample the number of distinct miRNAs with a read count above 0 and above 10 is plotted to give an impression about the abundance of distinct miRNAs and their read counts in the samples.\n\nThe number of identified miRNAs with either a read count above 0 (red) or 10 (green) is plotted for each sample.\n\nHeatmaps, PCA and t-SNE plots\n\nThe heatmaps, PCA and t-SNE plots are part of the unsupervised clustering methods that are applied by the miND pipeline and included in the report. For better understanding of underlaying group relationships, any grouping information available in the meta data file will be included in the graphs. Two heatmaps are generated in the interactive HTML report. The first includes only the top 50 miRNAs based on the coefficient of variation (see Figure 6) while the second one contains all miRNAs that were detected in all samples. Both heatmaps are based on RPM normalized reads and scaled using the unit variance method for visualization. Clustering is based on complete clusters using Euclidean distances as these methods are applicable for most experimental setups. The group association of each sample is shown in the heatmaps with colored bars at the top to visualize clustering of samples based on the provided grouping information. Multiple groups are supported for heatmaps (no groups limit) and PCA/t-SNE (maximum of two groups are shown by colors and shapes).\n\nGroup information provided with the experiment meta data XLS file is included if available.\n\n\nConclusions\n\nThe miND pipeline was developed and optimized for a multitude of small RNA-seq studies. While other available tools focus on specific aspects of the analysis (e.g., miRDeep2 on quantification of miRNAs and annotation of possible novel miRNAs and sRNAbench on differential expression), miND generates an extensive and standardized report suitable for the discovery phase of biomarker studies. The prepared HTML report provides a solid basis for further research and communicates the most important results in a structured and accessible way. Especially parameters relevant to quality control of the whole sequencing experiment (from library preparation to the in-silico analysis) are reported in standardized formats, to allow for a reliable and quick analysis of the overall quality of the experiment.\n\nBesides the results, the generated HTML report includes descriptions, hints, and details about the methods used. This ensures that the results can be interpreted and understood easily by non-statisticians or bioinformaticians. In addition, it ensures that the final HTML report contains all information needed for reproducibility and documentation of the analysis.\n\nData input and experimental setup of the miND pipeline can be adjusted with the given meta data file, making it possible to use the miND pipeline for various species, sample matrices and library preparation protocols.\n\nWith the availability of the source code of the pipeline under the GNU General Public License, additional analysis steps can be integrated into the R markdown report if needed, allowing the pipeline to be tailored to other specialized applications.\n\nWhile the miND pipeline includes an extensive set of analysis often needed in early phases of biomarker discovery studies, it is important to highlight the fact that no standardized pipeline will be sufficient and flexible enough to be used exclusively for every study. The results generated are meant to be a starting point for further analysis and optimizations, as parameters. For example, differential expression or heatmaps are chosen to give good results in most use cases but might not be the optimal for an individual project.\n\nThe miND pipeline was developed as part of the Translational Safety Biomarker Pipeline (TransBioLine) project from the IMI2 consortium. This project focuses on the discovery of miRNAs as novel biomarkers in the context of drug safety. In this case, the miND pipeline provides a standardized but still extensive first analysis of NGS data. In addition, the miND pipeline includes an extra module for the implementation of miND spike-ins for absolute quantification of microRNAs as recently published by Khamina et al. (2022).\n\nIn another recently published article by Gutmann et al. (2021) the pipeline was used in the discovery phase of the study to identify miRNAs that are associated with COVID-19 severity and mortality. The miRNAs reported by the miND pipeline were later manually selected and evaluated based on the HTML report for further confirmation with RT-qPCR, where the confirmation showed a high level of reproducibility from the NGS data.\n\nWe will continue working on the pipeline and release updates to the public version if needed. Especially in regard to the miND spike-ins that allow for the absolute quantification of miRNA in biofluids we expect to release an updated version soon.\n\n\nData availability\n\nMature and hairpin sequences of miRBase are available at: https://www.mirbase.org/ftp/22.1\n\nGenome sequences (DNA and cDNA) is available at Ensembl (for human): http://ftp.ensembl.org/pub/release-105/fasta/homo_sapiens\n\nNon-coding RNA sequences are available at RNAcentral: http://ftp.ebi.ac.uk/pub/databases/RNAcentral/current_release\n\nData associated with the example use case are not owned by the authors. Requirements to access these datasets is given in the protocol (https://dx.doi.org/10.17504/protocols.io.b3f6qjre).\n\n\nSoftware availability\n\nSource code available from: https://github.com/tamirna/miND\n\nArchived source code available from: https://doi.org/10.5281/zenodo.6080470 (Diendorfer et al., 2022)\n\nLicense: GNU GPL 3.0", "appendix": "References\n\nAnaconda Software Distribution: Anaconda Documentation. Anaconda Inc.; 2020. Reference Source\n\nAndrews S: FastQC: A quality control tool for high throughput sequence data. 2010Reference Source\n\nAparicio-Puerta E, Lebrón R, Rueda A, et al.: sRNAbench and sRNAtoolbox 2019: intuitive fast small RNA profiling and differential expression. Nucleic Acids Res. 2019; 47(W1): W530–W535. PubMed Abstract \| Publisher Full Text\n\nChen Y, Lun ATL, Smyth GK: From reads to genes to pathways: Differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline [version 2; referees: 5 approved]. F1000Res. 2016; 5: 1–49. Publisher Full Text\n\nde Ronde MWJ , Ruijter JM, Moerland PD, et al.: Study Design and qPCR Data Analysis Guidelines for Reliable Circulating miRNA Biomarker Experiments: A Review. Clin. Chem. 2018; 64(9): 1308–1318. PubMed Abstract \| Publisher Full Text\n\nDiendorfer A, Khamina K, Pultar M, et al.; miND (miRNA NGS Discovery pipeline): a small RNA-seq analysis pipeline and report generator for microRNA biomarker discovery studies (v1.2RC2). Zenodo. 2022. Publisher Full Text\n\nEwels P, Magnusson M, Lundin S, et al.: MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016; 32(19): 3047–3048. PubMed Abstract \| Publisher Full Text\n\nFriedländer MR, MacKowiak SD, Li N, et al.: MiRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res. 2012; 40(1): 37–52. PubMed Abstract \| Publisher Full Text\n\nGriffiths-Jones S: The microRNA registry. Nucleic Acids Res. 2004; 32(Database issue): 109D–1111D. PubMed Abstract \| Publisher Full Text\n\nGrüning B, Dale R, Sjödin A, et al.: Bioconda: sustainable and comprehensive software distribution for the life sciences. Nat. Methods. 2018; 15(7): 475–476. PubMed Abstract \| Publisher Full Text\n\nGutmann C, Khamina K, Theofilatos K, et al.: Association of cardiometabolic microRNAs with COVID-19 severity and mortality. Cardiovasc. Res. 2021; 118: 461–474. PubMed Abstract \| Publisher Full Text\n\nKhamina K, Diendorfer AB, Skalicky S, et al.: A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) for Genome-Scale Analysis and Absolute Quantitation of Circulating MicroRNA Biomarkers. Int. J. Mol. Sci. 2022; 23(3): 1226. PubMed Abstract \| Publisher Full Text\n\nKöster J, Rahmann S: Snakemake-a scalable bioinformatics workflow engine. Bioinformatics. 2012; 28(19): 2520–2522. Publisher Full Text\n\nLangmead B, Trapnell C, Pop M, et al.: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3): R25. PubMed Abstract \| Publisher Full Text\n\nLarrea E, Sole C, Manterola L, et al.: New Concepts in Cancer Biomarkers: Circulating miRNAs in Liquid Biopsies. Int. J. Mol. Sci. 2016; 17(5): 627. PubMed Abstract \| Publisher Full Text\n\nLee RC, Ambros V: An Extensive Class of Small RNAs in Caenorhabditis elegans. Science. 2001; 294(5543): 862–864. PubMed Abstract \| Publisher Full Text\n\nLlewellyn HP, Vaidya VS, Wang Z, et al.: Evaluating the Sensitivity and Specificity of Promising Circulating Biomarkers to Diagnose Liver Injury in Humans. Toxicol. Sci. 2021; 181(1): 23–34. PubMed Abstract \| Publisher Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 521–550. PubMed Abstract \| Publisher Full Text\n\nMölder F, Jablonski KP, Letcher B, et al.: Sustainable data analysis with Snakemake. F1000Res. 2021; 10: 33. Publisher Full Text\n\nMurillo OD, Thistlethwaite W, Rozowsky J, et al.: exRNA Atlas Analysis Reveals Distinct Extracellular RNA Cargo Types and Their Carriers Present across Human Biofluids. Cell. 2019; 177(2): 463–477.e15. PubMed Abstract \| Publisher Full Text\n\nO’Brien J, Hayder H, Zayed Y, et al.: Overview of microRNA biogenesis, mechanisms of actions, and circulation. Front. Endocrinol. 2018; 9(AUG): 1–12. PubMed Abstract \| Publisher Full Text\n\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009; 26(1): 139–140. PubMed Abstract \| Publisher Full Text\n\nRonen R, Gan I, Modai S, et al.: miRNAkey: a software for microRNA deep sequencing analysis. Bioinformatics. 2010; 26(20): 2615–2616. PubMed Abstract \| Publisher Full Text\n\nStarlinger P, Hackl H, Pereyra D, et al.: Predicting Postoperative Liver Dysfunction Based on Blood-Derived MicroRNA Signatures. Hepatology. 2019; 69(6): 2636–2651. PubMed Abstract \| Publisher Full Text\n\nSweeney BA, Petrov AI, Burkov B, et al.: RNAcentral: A hub of information for non-coding RNA sequences. Nucleic Acids Res. 2019; 47(D1): D221–D229. PubMed Abstract \| Publisher Full Text\n\nWang W-C, Lin F-M, Chang W-C, et al.: miRExpress: Analyzing high-throughput sequencing data for profiling microRNA expression. BMC Bioinformatics. 2009; 10(1): 328. PubMed Abstract \| Publisher Full Text\n\nZerbino DR, Achuthan P, Akanni W, et al.: Ensembl 2018. Nucleic Acids Res. 2018; 46(D1): D754–D761. PubMed Abstract \| Publisher Full Text" }	[ { "id": "129126", "date": "14 Apr 2022", "name": "Kristian Almstrup", "expertise": [ "Reviewer Expertise Endocrinology", "small RNAs", "genetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript by Diendorfer et al., a bioinformatic pipeline for analysis of data from small RNA sequencing is presented. The pipeline, named miND, allows identification and annotation of small RNA reads as well as differential expression analysis.\nI have the following major concerns about the study as it is:\nSeveral other small RNA sequencing pipelines, like Oasis2.0 (https://oasis.dzne.de/index.php), sRNAWorkbench (https://sourceforge.net/projects/srnaworkbench/), sRNAPipe (https://github.com/GReD-Clermont/sRNAPipe), miRge3.0 (https://sourceforge.net/projects/mirge3/) already exist and some support both identification and differential expression analysis. It is hence unclear what novelty miND brings compared to other pipelines. To allow the reader to make an informed choice about which pipeline to choose for analysis, miND should be benchmarked against some of the already existing pipelines. What are the differences when the same dataset (PRJEB27261/E-MTAB-6885) is analysed with e.g. Oasis2.0 (Rahman et al., 2018)?\nThe authors argue that miND “bridges the gap between biologists and bioinformaticians”, and this is also evident from the easy-to-use Excel files. However, the pipeline is based on Snakemake workflows and a conda install and hence require a priori knowledge of conda, which would not be common knowledge to biologists. I encourage the authors to make miND available as a standalone app or web portal (as is the case for the similar pipeline Oasis2.0). Since, at least, parts of the miND pipeline are based on R-scripts it might be easy to make a Shiny app or similar.\nThe pipeline focuses on miRNAs. This reviewer encourages the authors also to include analysis of other small RNA species as these are likely to be equally important as biomarkers in a liquid biopsy. Furthermore, on a whole, the authors do not discuss in detail the possible problems and downsides of their pipeline. A more in-depth and critical discussion of strengths and limitations is warranted.\nThe readme file on github should contain instructions on how to install miND in a language that biologists can understand.\nFinally, in some places the authors should consider shortening sentences/simplifying statements so, again, it is easier for the readers to follow.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "151316", "date": "05 Oct 2022", "name": "Francisco J. Enguita", "expertise": [ "Reviewer Expertise non-coding RNAs", "miRNAs", "lncRNAs", "circRNAs", "structural biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Diendorfer and coworkers describes a pipeline for NGS data processing specially devoted to the analysis of small non-coding RNAs, mainly focused on miRNAs.\nThe manuscript is well written, but the authors would need to give further details in order to compare their pipeline with the already existing ones. It is not clear for the reader what are the main advantages of miND pipeline in comparison with the already available ones.\nI would advise to perform a small benchmarking study using a test dataset that could be any one existing in public databases. The authors would need to answer the questions: what are the advantages of miND, its weaknesses and why the user should give a try to this new pipeline.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] } ]	1	https://f1000research.com/articles/11-233
https://f1000research.com/articles/11-230/v1	24 Feb 22	{ "type": "Opinion Article", "title": "Education research is still the hardest science: a proposal for improving its trustworthiness and usability", "authors": [ "Gustavo Fischman", "Audrey Amrein-Beardsley", "Stephanie McBride-Schreiner", "Audrey Amrein-Beardsley", "Stephanie McBride-Schreiner" ], "abstract": "In this essay, we argue that colleges of education, particularly those at research-intensive institutions, favor simplistic notions of scholarly impact and that this trend has concerning implications for the field, for researchers, and for the public at large. After describing the challenges and shortcomings of the current models of research assessment in education, we outline an alternative proposal in which trustworthiness and usability of research would complement traditional metrics of scholarly relevance. This proposal encourages a twofold approach to research assessment that involves (1) a more thorough analysis of the limitations and problems generated by the use of simplistic notions of scholarly impact, and (2) a commitment to the implementation of more equitable systems based on a broader range of assessment measures to assess faculty research contributions.", "keywords": [ "colleges of education", "research impact", "research assessment" ], "content": "Introduction\n\nTwo decades ago, David C. Berliner (2002) warned of the risks involved when research funding policies are based on narrow definitions of what acceptable science is. He argued that:\n\n“Hard-to-do science is what the social scientists do and, in particular, it is what we educational researchers do. In my estimation, we have the hardest-to-do science of them all! We do our science under conditions that physical scientists find intolerable. We face particular problems and must deal with local conditions that limit generalizations and theory building—problems that are different from those faced by the easier-to-do sciences.” (p. 18)\n\nToday, government funding agencies still give preference in education to “easier-to-do sciences” when it comes to research methods. The good news, however, is that they no longer explicitly exclude multiple approaches for conducting research in education. The bad news is that the field of education research itself has adopted a very narrow set of indicators for judging what is acceptable as good research.\n\nAs faculty, researchers, scholars, and editors working in higher education, we see an unsettling trend. Measures of impact—social, global, and real-world—are increasingly expected of scholarly research; yet, the assessment of these outcomes remains vague, arbitrary, and one-size-fits-all across disciplines. Further, the scholarly publishing ecosystem, which produces the most revered and measurable indicators of scholarly impact and innovation, has grown more commercialized and profit driven, leading to an ever wider disconnect between the producers of scholarly knowledge (e.g., researchers, funding agencies, and the community at large) and the conveyors of that knowledge (e.g., academic publishers). We argue that those in higher education in general, but colleges of education in particular, need to discuss and challenge simplistic notions of scholarly impact and move towards a more trustworthy and informed culture and infrastructure of scholarly assessment.\n\nA common understanding of scholarly impact is often related to two indicators: number of articles in flagship journals and number of citations (Aguinis et al., 2012). For many scholars and organizations (Anderson et al., 2021; Hicks et al., 2015; Walker, 2017), however, such definitions of impact, especially when institutionalized in college standards or university tenure and promotion policies and procedures, are not only inadequate to assess scholarly relevance, but also embody the wrong kind of incentives.\n\nConsider the following scenario. A professor with research expertise in high-interest topics writes an insightful blog with thousands of non-academic followers. When evaluated for promotion, the professor is told by senior faculty not to mention the blog given its lesser academic status, despite the fact that education influencers like Diane Ravitch, Frederick Hess, and Mercedes Schneider have enormous blog audiences with millions of page views each.\n\nTake another example. A professor, who is frequently consulted by research organizations in other countries about implementing good practices in scholarly publications and the organization of academic events is advised that for promotion purposes only collaborations with reputable United States (U.S.)-based organizations will be considered. How can such activities, which clearly build on these professors’ research and expertise, increase the recognition and prestige of the professors and their affiliated institutions, disseminate relevant knowledge without barriers, and offer evidence of significant reach and contribution to the public good, be viewed as so marginal in terms of scholarly impact?\n\nAnother often overlooked and misunderstood dimension of the assessment of scholarly impact is the structure and culture of scholarly publishing. For example, a journal might be consulted about its impact to help decide a tenure and promotion case at a university. All members but one on the review panel recommend tenure. The hold-out states that the author did not publish in enough high impact journals. One of the journals in question is an open access (OA) publication recognized as influential by many scientific organizations, with thousands of academic and non-academic readers worldwide and a solid record of citations per article, but due to its nontraditional publishing structure and multilingual nature, it was denied a journal impact factor by the publishing affiliate that assigns such measures. How could such a journal be excluded from recognized (albeit imperfect) indicators of impact and then, because of that peculiarity, be branded as not high impact when article-level and engagement metrics suggest otherwise?\n\nHere we propose that the field of education research moves away from the imperfect and ineffective notion of impact and similar terms like “returns”, “benefits” and “value”, and toward more comprehensive and field-specific scholarly assessment strategies. In the next section, we focus on the challenges and shortcomings of the current models of assessment of research impact in education, and then outline a proposal in which the trustworthiness and usability of research would complement current metrics of scholarly relevance.\n\n\nIs impact a new fetish of education research?\n\nIn the U.S. and globally, colleges of education, primarily at research intensive universities are converging on the idea that evidence of impact is of utmost importance. Impact has become a new fetish (Wood, 2021). This increased fascination with finding better indicators of scholarly impact and influence relies on formulaic uses of metrics-based reward and punishment assessment processes to accomplish three simultaneous and elusive goals: increase research impact, enhance institutional prestige, and demonstrate high levels of scholarly productivity and innovation (Schneider, 2015).\n\nToday it is rare to find colleges of education that are not requesting faculty to provide annual evaluative reports with measurable metrics, such as numbers of articles published in “High Impact Factor Journals,” numbers of citations per article, and other indicators of impact (e.g., Google Scholar’s h-indices, Publish or Perish scores, levels of engagement in Kudos, the RG Score from Research Gate). Also pertinent in judging the quality of a faculty member’s work are indicators such as publications in journals with high rejection rates or sponsored by esteemed professional associations (e.g., American Educational Research Association [AERA]; publications by university presses (e.g., Oxford University Press); and funds, grants, and research awards bestowed by organizations (e.g., Institute of Education Sciences [IES]).\n\nThis model of holding university professors accountable for their presumed impact, or rather the impact of their scholarly products and dossiers, is not new (Boyer, 1996; Weiss, 1981), nor exclusive to colleges of education, however. Many fields are subject to the “metric tide” (Wilsdon et al., 2015) and “ranking mania” (European University Association, 2013, p. 6) that increasingly frames the assessment and evaluation policies and procedures of contemporary universities. As Shewchuk and Cooper (2018) concluded, after conducting an analysis of 721 indicators of research impact for social sciences in 32 countries:\n\n“What is clear from the veritable explosion of research impact materials in the past decade and the increasing number of performance-based research funding systems arising globally is that research impact will be a defining factor of research infrastructure, funding and landscapes across the world for the foreseeable future.” (p. 63)\n\nWe do not oppose the use of clear indicators and metrics to assess research and defend the principle of scholars’ curiosity as driver of scientific endeavors. We also do not support a nostalgic return to evaluation systems used during idealized eras of universities governed by autonomous communities of scholars. We do, however, believe education researchers need to be more cautious and identify, resist, and replace assessment policies based on poorly constructed and misleading metrics that will not improve education research, nor its usability, relevance, and value.\n\n\nCan we assess scholarly contributions in education without being simplistic?\n\nWhat is research impact? There is a distinction between academic impact, understood as the intellectual contribution to one’s field of study within academia, and external socio-economic impact, effects beyond academia (Penfield et al., 2014). Impact is multifaceted, dynamic, temporal, and not always beneficial. Meanings and judgments of impact differ across disciplines and vary as cultures, policies, and contexts change.\n\nFew would dispute the claim that the impact of education research is both elusive and subjective. As Kaestle (1993) noted in The Awful Reputation of Education Research, the goal to increase the reputation and impact of educational scholarship has deep roots: “[I] f education researchers could reverse their reputation for irrelevance, politicization, and disarray […] they could rely on better support because most people, in the government and the public at large, believe that education is critically important” (pp. 30–31).\n\nSome educational researchers have attempted to collaborate with practitioners to yield more impactful research to address problems in real classrooms, schools, and universities (Penuel et al., 2016). Yet, as Berliner (2002) underscored in the opening quote, due to the varied and complex nature of education systems, education research is contextual. Types of research also matter. Current impact indictors favor research with more immediate visible results over other types of research with less immediate or tangible impact (Laing et al., 2018).\n\nColleges of education, then, face a conundrum. Despite ample consensus regarding the desirability of producing more studies with the explicit purpose of improving education (Penuel et al., 2016), and more broadly focused research oriented to the public good, no effective and fair system exists that captures the full picture of the relevance and impact of scholarship in a field as diverse as education (Anderson et al., 2021; Simons, 2008).\n\nConsequently, in most cases instead of adopting contextualized and measured models, many colleges of education adopt overly simplified systems of impact assessment based on indirect measures of scholarly relevance such as the Journal Citation Record (JCR) from Scopus, and the Journal Impact Factor (JIF), published Web of Science. These measures have been long been controversial (Alperin et al., 2019; Simons, 2008) yet convey a sense of being purely meritocratic, by using appropriate indicators of impact, relevance, and influence (Fischman, 2016; Zuiker et al., 2019). In other words, the indicators easiest to quantify may or may not promote the most impactful education research. Such metrics are developed using sophisticated algorithms that yield robust statistics to be consumed and trusted, and also ranked, categorized, monetized, regardless of the validity of the inferences derived. This phenomenon, the “simplimetrification of educational research” (Fischman, 2016), has the ironic effect of allowing researchers and their institutions to feel good about themselves, by confusing continuous increases of countable items (e.g., the more articles and more citations in more exclusive journals) with substantial scientific and pedagogical contributions.\n\n\nThe academic publishing dimension\n\nThis metric tide, in conjunction with the publish or perish imperative, has generated a veritable tsunami with gigantic waves of articles that follow pre-established, tidy paths of exploration that may be accurately measured and rewarded. The publication of educational research, however, is not completely altruistic or disinterested. Academic publishers and editors are keen to prove that the research they publish is influential in order to attract new submissions and subscriptions. To attract funding and prospective students, researchers and administrators are keen to prove that the research they produce is influential. Both the JCR and the JIF—assigned to journals, not individual articles—are attractive, recognizable metrics that conflate journal performance with individual researcher performance, thus serving the aforementioned, multiple interests simultaneously.\n\nAs Puehringer et al. (2021) noted, the political economy of academic publishing entails “publishers sell a highly profitable, yet immensely publicly subsidized product” (p. 2). Academic publishing is a vast, lucrative industry, with revenues estimated at USD 26 billion (Johnson et al., 2018). The rising demand for free and digital access to research over the last 30 years led commercial publishers to adopt hybrid models that balance traditional practices (e.g., via subscriptions and paywalls) with new OA schemes that offset publishing costs by charging researcher-authors—often paid out of research accounts provided by institutions or funders—to make published content freely available. Further, academic libraries broker serial deals with commercial publishers to access the same content through subscription bundles, essentially buying back access to the research that the researchers at their institutions produced (Wenzler, 2017). The ironic result is that much potentially impactful research is hidden away behind paywalls because many authors choose not to, or are unable to pay the fees to make their research available to all.\n\nLinked to these financial aspects, scholarly publication formats (print, digital, hybrid) and types (subscription-based, OA, etc.) are complex. Further, multiple OA publishing options exist, including but not limited to Gold OA, Delayed OA, Green OA, and Platinum or Diamond OA. Adding to this complexity, OA articles have a range of copyright licenses with varying degrees of permissions. A lack of awareness among researchers about the differences between publication types and associated licensing leads to the general misconception that all OA publications are free, which is untrue. All publications have a cost; the difference is who pays (readers, authors, institutions, libraries, funders, publishers, etc.)\n\nInequities embedded in the scholarly publishing landscape, such as biases for English-language works (e.g., more than 33% scientific documents on global conservation are published in languages other than English but are critically ignored; see, e.g., Amano, 2021), also have implications for research assessment that are frequently overlooked (Kubota, 2020). The circularity of these biases, stemming from the Western market-oriented nature of scholarly publishing, are striking. For example, a journal article indexed in Scopus or Web of Science is viewed as an indicator of research quality and international reach (Sivertsen, 2016, p. 357). Journals registered in these influential indexes are more likely to publish English-only articles, given the editorial boards and editors also conduct their activities in English (Vasen & Vilchis, 2017). Moreover, a journal article published in U.S.-based Scopus or UK-based Web of Science is more likely to have a JIF, also assigned by Web of Science. In fields like the social sciences, journals with high JIFs tend to have higher APCs, potentially excluding submissions by authors from less affluent countries, who are not native English speakers, or both (Demeter & Istratii, 2020, p. 506). Considering all of the above, in some subject areas, the correlation between high APCs and JIF and JCR, combined with the existing economic inequalities among countries, reinforces existing hierarchies of language as maintained by publisher databases.\n\nWhile no scholarly or business enterprise is perfectly equitable, such biases and circularity—and their reinforcement through academic research assessment processes—are highly concerning. Commercial publishers capitalize on the decentralized, siloed nature of academic institutions and research communities (who are also in competition for research dollars and rankings) and a lack of in-depth knowledge about the scholarly publishing process. Researchers should be wary of giving up more control over who is defining and measuring research quality and impact (Aspesi & Scholarly Publishing and Academic Resources Coalition [SPARC], 2019). Given that quality, levels of international engagement, and societal relevance certainly should be promoted in research assessment, should coverage by a commercial indexing service be a criterion for research quality or an indicator of global engagement?\n\n\nResisting the metric tide in education scholarship: trustworthiness and usability\n\nIn our view, this model rewards people based on metrics and measurements that do not differentiate between research articles concluding with the statement “more research is needed” and those that bring value to a scholarly field, help educators improve their practice, or supply compelling evidence to policymakers for important decisions. Rather, education researchers learn new terms and tools about scholarly assessment, instead of expanding curious research, asking better and more relevant questions for the advancement of the field, or producing more usable knowledge. Unfortunately, this fascination with simpler models, combined with a disconnect from the publishing ecosystem, ultimately lead researchers to an uncritical and sometimes naïve acceptance of the accuracy and explanatory power of these indicators.\n\nIn recent years, a number of initiatives have emerged to push against this tide of simplimetrification, as groups of researchers have converged to develop guidelines for research evaluation and assessment without using one-dimensional measures. Some prominent examples include the Leiden Manifesto (http://www.leidenmanifesto.org/), the San Francisco Declaration on Research Assessment (DORA; https://sfdora.org/), the Panama Declaration of Open Science (https://web.karisma.org.co/declaraciondepanama/), and the Hong Kong Principles (https://www.wcrif.org/guidance/hong-kong-principles). Since 2019, the Hong Kong Principles, for example, have promoted research assessment based on five key tenets: responsible research practices, transparent reporting, open science (open research), valuing diverse types of research, and recognizing all contributions to scholarly activity. Collectively, these researcher-led activities represent pushback against an unbalanced system of research assessment in which individual researchers face a “one-sided emphasis on traditional, quantifiable output indicators,” despite the fact that “bibliometric indicators tell a story, but not the whole story” (Dutch Research Council, 2019, p. 4).\n\nSuch recommendations are, accordingly, gaining traction, and advocates are moving words into action. In 2019, Consejo Latinoamericano en Ciencias Sociales (CLACSO; https://www.clacso.org/) organized FOLEC—Foro Latinoamericano sobre Evaluación Científica (Latin American Forum of Scientific Evaluation)—to develop better systems of assessment consistent with Open Science principles. The metric tide is turning in Asia, as shown by the Chinese government’s decision to stop using the JIF and similar indirect metrics as the key indicator of research assessment (Zhang & Sivertsen, 2020). Also noteworthy, the European Research Council decided to disallow mention of indirect journal metrics in research funding applications (Matthews, 2021). The call by Dutch universities and funding agencies for a revamped system of recognition and rewards, based on diverse talent, academic interdependence, emphases on quality over quantity, and the encouragement of open science and high-quality leadership, captures the essence of these initiatives (Dutch Research Council, 2019, p. 3).\n\n\nShould trustworthiness and usability be considered in assessing scholarship in education?\n\nBuilding on the heavy lifting of those mentioned in the previous section, we propose that a better way to assess education research requires a combined use of existing indicators and metrics with evidence of enhanced trustworthiness and usability—within and beyond disciplinary, professional, or technical communities—to foster and sustain processes of conceptual inquiry and education problem solving. As per the aforementioned Hong Kong Principles, “The primary goal of research is to advance knowledge. For that knowledge to benefit research and society, it must be trustworthy. Trustworthy research is robust, rigorous and transparent at all stages of design, execution and reporting” (Moher et al., 2020, p.1).\n\nTrustworthiness is not a given and not an eternal quality (Schwandt et al., 2007). Robust findings may be trustworthy in one decade and not another. As the group Science in Transition (Dijstelbloem et al., 2013) pointed out, researchers must also address the increasing mistrust from the public about scientific expertise and tell the public how science really works. Trust in the results of education research, no matter how rigorous the procedures used, is never simply assumed. Trusting the process and results of any research will always involve moral, cultural, and political considerations (Little & Green, 2021). To increase the trustworthiness of education research, it is necessary, yet not sufficient, to provide wide access to the knowledge produced and engagement with the ideas and data derived from such research.\n\nAccess to and engagement with scholarship entail more than depositing knowledge in the library or an OA journal, book, or repository. These matters also rest on other aspects of research, such as language, previous knowledge of the phenomenon studied, ideological preferences, and the like (Suber, 2016). Regarding access, how easy might it be to access the knowledge produced and what might be the barriers to accessing that knowledge? Did the knowledge reach its intended audiences (e.g., scholars, professionals, policy makers, or practitioners in the field)? Did the knowledge reach general, non-targeted audiences? Regarding engagement, to what extent do the central ideas, procedures, data, and conclusions enter into our systems of knowledge exchange with our intended audiences? In other words, if the research is not accessible due to various barriers (e.g., language, technology, disability, paywalls, and the like) then how can it be considered trustworthy by its intended audiences?\n\nEducation research will not be usable unless it is trusted; thus, trustworthiness and usability are inexorably linked. By usability we mean processes that signal potential access and engagement by both specialists as well as practitioners, each group accessing and engaging on their own terms, in their own time, and according to their own needs. This notion of usability also requires access and engagement with five critical components of knowledge generated by research: learnability, efficiency, memorability, integrity, and satisfaction derived via the knowledge produced (Han et al., 2001).\n\nIn our understanding, usability is not a measure of dissemination or implementation, nor a description of processes or products. Here, we want to emphasize that we are not advocating for usefulness as a key indicator of relevance as others have done (Buckhardt & Shoenfeld, 2003). We welcome research that has direct applications in teaching and learning. But defending the principle that practical and immediate implementation is not, and should not be, the goal of all education research. Conceptual studies directed at understanding and developing theories, for example, could prove very relevant and usable. What we propose, instead, is that at the institutional level education research should be promoted, and thus incentivized and assessed considering its usability, not only in the abstract form of the well-known questions of “So what?” and “Who cares?” but also in concrete steps taken to support strategies that help researchers mobilize research results.\n\nThe condition that we want to underscore is that trustworthiness and usability are not intrinsic qualities of the knowledge derived from any research endeavor, but characteristics that require intentional strategies that need to be incentivized to be implemented. We agree with others (viz., Berliner, 2002; Campbell et al., 2017; Hess, 2008) who warned about the shortcomings of reducing education research to methodological or technical matters. Improving access and engagement and opening diverse dialogues among researchers, policymakers, practitioners, and the public demand close attention to techniques and methods, but an even closer engagement with what is ethically, politically, and pedagogically desirable. As scholars, these desirable outcomes are linked to greater opportunities for open, interdisciplinary, and intersectional inquiries, welcoming a plurality of epistemic standpoints, and strengthening the commitment to contribute to the public good. Next, we ask what can be done to encourage more comprehensive assessments of education research.\n\n\nWhat can be done?\n\nPerhaps the first step is to interrogate and challenge the notion that the simplimetrification of assessing research in education is unavoidable. Understanding its administrative advantages as systems of distributions of rewards and punishments, as well as acknowledging its shortcomings, is the first step toward more trustworthy and usable research in education.\n\nIf we reconsider the opening vignettes in light of a new model based on trustworthiness and usability, quite a different story of assessment unfolds. Professors writing a blog with thousands of primarily non-academic followers would be supported and recognized in their efforts to interact with the public and build trust in scholarship through blogging. Professors consulted by numerous international research organizations in other countries would be supported and recognized for their contributions to global engagement and support of multilingualism within scholarly communications, both of which foster trust in research between U.S.-based or non-U.S.-based research communities.\n\nOne potential path to an assessment system based on trust and usability is a renewed commitment to the raison d'être of education research: its pedagogical function. As the editors of the British Journal of Educational Research argued, the field needs to combine the search for identifying and posing education problems with inquiries that pose solutions to those same problems:\n\n“Educational research that operates in a problem-posing rather than a problem-solving mode is, in this regard, not just research on or about or for education, but is, in a sense, itself a form of education as it tries to change mindsets and common perceptions, tries to expose hidden assumptions, and tries to engage in ongoing conversations about what is valuable and worthwhile in education and society more generally.” (Biesta et al., 2019, p. 3)\n\nWe believe that for this type of education research to be more widespread, colleges of education, accordingly, need to complement the use of indirect indicators of scientific rigor with evidence of efforts to increase trustworthiness and usability. To foster such an approach, we ask those conducting, publishing, and assessing education research to consider doing the following:\n\n1) Avoid simplistic models: Following the lead of the Open Science movement, DORA and others, all while considering complementing indirect measures of “impact” in assessment activities with more nuanced indicators of the quality, usability, and trustworthiness of a wide range of research products.\n\n2) Avoid one-size-fits-all approaches: The usability of education research cannot be reduced to how practical or applied knowledge is, but to what extent it is potentially accessible to other researchers, stakeholders, and users (e.g., practitioners, policymakers, journalists, and the public).\n\n3) Engage with field-specific models: Adopt and advocate for expanded indicators within systems of research assessment that are specifically relevant to other scholars in the field, as well as practitioners, policymakers, journalists, and the public.\n\n4) Offer institutional support: The trustworthiness and usability of research need to be earned through interactive processes promoted and sustained institutionally. Individual scholars are trained to do good research and spend considerable effort in the analyses and syntheses of data, reviewing manuscripts, presenting at conferences, and the like. Making our results more usable and increasing trustworthiness requires time and effort in the form of producing complementary materials (e.g., podcasts, blogs, op-eds, video-commentaries, policy briefs, workshops). Colleges of education would greatly benefit and reduce some of the inequalities derived relatively simplistic models by allocating resources to increase the collective relevance of research production.\n\n5) Account for context, language, and time: It is impossible to forecast the trajectory of scholarship, whereby the usability and trustworthiness of education research depends on the context of production, the languages used, as well as the time and timeliness of a publication.\n\n6) Promote and reward efforts to remove barriers to research access and use: Recognize that quite a bit of very good scholarship is published in OA journals, as well as raise the awareness of the complexity of this model. Supporting OA publishing for researchers with limited resources, such as students, early career scholars, and scholars working in languages other than English, are also worthy ventures.\n\nA first, though not an easy, move away from this unfair and ineffective system is to recognize alternatives and redirect our debates beyond the important, yet insufficient, question: How influential is the placement of a research contribution (e.g., article, book, or chapter) on the assessment of the merit of a scholar? Instead, we must embrace more comprehensive, and field specific systems of incentives and assessment oriented to the production of scholarship that contributes to the public good, encourages collaboration, and promotes interdisciplinary and intersectional research, and endeavors to increase access, trustworthiness, and responsiveness to both practical demands as well as conceptual challenges. As education researchers, our responsibility to avoid easy-to-implement models of scholarship assessment that end up producing more research that matters less.\n\n\nData availability\n\nThere are no underlying data associated with this article.", "appendix": "References\n\nAguinis H, Suárez-González I, Lannelongue G, et al.: Scholarly impact revisited. Acad. Manag. Perspect. 2012; 26(2): 105–132. Publisher Full Text\n\nAlperin JP, Nieves CM, Schimanski LA, et al.: Meta-research: How significant are the public dimensions of faculty work in review, promotion and tenure documents?. elife. 2019; 8: e42254. PubMed Abstract \| Publisher Full Text\n\nAmano T: The English language dominates global conservation science – which leaves 1 in 3 research papers virtually ignored. The Conversation. 2021. Reference Source\n\nAnderson G, Gray-Nicolas NM, Payton M: Education faculty as knowledge brokers: Competing for access to New York State print media and policy influence. Education Policy Analysis Archives. 2021; 29(12). Publisher Full Text\n\nAspesi C; Scholarly Publishing and Academic Resources Coalition (SPARC): About the Report. Landscape analysis. 2019, March 29. Reference Source\n\nBerliner D: Educational research: The hardest science of all. Educ. Res. 2002; 31(8): 18–20. Publisher Full Text\n\nBiesta G, Filippakou O, Wainwright E, et al.: Why educational research should not just solve problems, but should cause them as well. Br. Educ. Res. J. 2019; 45(1): 1–4. Publisher Full Text\n\nBoyer EL: From scholarship reconsidered to scholarship assessed. Quest. 1996; 48(2): 129–139. Publisher Full Text\n\nBurkhardt H, Schoenfeld AH: Improving educational research: Toward a more useful, more influential, and better-funded enterprise. Educ. Res. 2003; 32(9): 3–14. Publisher Full Text\n\nCampbell C, Pollock K, Briscoe P, et al.: Developing a knowledge network for applied education research to mobilise evidence in and for educational practice. Educ. Res. 2017; 59(2): 209–227. Publisher Full Text\n\nDemeter M, Istratii R: Scrutinizing what open access journals mean for global inequalities. Publ. Res. Q. 2020; 36: 505–522. Publisher Full Text\n\nDijstelbloem H, Huisman F, Miedema F, et al.: 2013. Why science does not work as it should. And what to do about it. [Science in Transition Position Paper]. Reference Source\n\nDutch Research Council: Room for everyone's talent. [Position paper]. NWO; 2019. Reference Source\n\nEuropean University Association (EUA): Global university rankings and their impact: Report II. 2013. Reference Source\n\nFischman GE: The simplimetrification of educational research [Blog post]. Education International. 2016. Reference Source\n\nHan SH, Yun MH, Kwahk J, et al.: Usability of consumer electronic products. Int. J. Ind. Ergon. 2001; 28: 143–151. Publisher Full Text\n\nHess FM: When research matters: How scholarship influences education policy. Harvard Education Press; 2008.\n\nHicks D, Wouters P, Waltman L, et al.: Bibliometrics: The Leiden Manifesto for research metrics. Nature. 2015; 520: 429–431. PubMed Abstract \| Publisher Full Text\n\nJohnson R, Watkinson A, Mabe M: 2018. The STM report. An overview of scientific and scholarly publishing. International Association of Scholarly, Technical and Medical Publishers. Reference Source\n\nKaestle CF: The awful reputation of education research. Educ. Res. 1993; 22(1): 23–31. Publisher Full Text\n\nKubota R: Confronting epistemological racism, decolonizing scholarly knowledge: Race and gender in applied linguistics. Appl. Linguis. 2020; 41(5): 712–732. Publisher Full Text\n\nLaing K, Mazzoli Smith L, Todd L: The impact agenda and critical social research in education: Hitting the target but missing the spot?. Policy Futures Educ. 2018; 16(2): 169–184. Publisher Full Text\n\nLittle D, Green DA: Credibility in educational development: Trustworthiness, expertise, and identification. High. Educ. Res. Dev. 2021: 1–16. Publisher Full Text\n\nMatthews D: European Research Council bans journal impact factor from bids. Times Higher Education. 2021. Reference Source\n\nMoher D, Bouter L, Kleinert S, et al.: The Hong Kong Principles for assessing researchers: Fostering research integrity. PLoS Biol. 2020; 18(7): e3000737. PubMed Abstract \| Publisher Full Text\n\nPenfield T, Baker MJ, Scoble R, et al.: Assessment, evaluations, and definitions of research impact: A review. Res. Eval. 2014; 23(1): 21–32. Publisher Full Text\n\nPenuel WR, Briggs DC, Davidson KL, et al.: Findings from a national survey of research use among school and district leaders. National Center for Research in Policy and Practice; 2016. Reference Source\n\nPuehringer S, Rath J, Griesebner T: The political economy of academic publishing: On the commodification of a public good. PLoS One. 2021; 16(6): e0253226. PubMed Abstract \| Publisher Full Text\n\nSchneider J: Looking outside education: Expanding our thinking about moving research into practice. Educ. Policy Anal. Arch. 2015; 23(119). Publisher Full Text\n\nSchwandt TA, Lincoln YS, Guba EG: Judging interpretations: But is it rigorous? Trustworthiness and authenticity in naturalistic evaluation. N. Dir. Eval. 2007; 2007: 11–25. Publisher Full Text\n\nShewchuk S, Cooper A: Research impact, the ‘new academic capital’: An environmental scan of research impact indicators and resources for the humanities and social sciences across 32 countries. J. Soc. Sci. 2018; 14(1): 55–64. Publisher Full Text\n\nSimons K: The misused impact factor. Science. 2008; 322(5899): 165. PubMed Abstract \| Publisher Full Text\n\nSivertsen G: Patterns of internationalization and criteria for research assessment in the social sciences and humanities. Scientometrics. 2016; 107: 357–368. PubMed Abstract \| Publisher Full Text\n\nSuber P: Knowledge unbound: Selected writings on open access, 2002–2011. The MIT Press; 2016; 456.\n\nVasen F, Vilchis IL: Sistemas nacionales de clasificación de revistas científicas en América Latina: Tendencias recientes e implicaciones para la evaluación académica en ciencias sociales. Revista Mexicana de Ciencias Políticas y Sociales. 2017; 62(231): 199–228. Publisher Full Text\n\nWalker VS: La evaluación como mecanismo de regulación del trabajo académico. Estudio de casos en universidades de Argentina y España. Archivos Analíticos de Políticas Educativas. 2017; 25(108). Publisher Full Text\n\nWeiss CH: Measuring the use of evaluation. Ciarlo JA, editor. Utilizing evaluation: Concepts and measurement techniques. SAGE; 1981; (pp. 17–33).\n\nWenzler J: Scholarly communication and the dilemma of collective actions: Wy academic journals cost too much. C&RL. 2017; 78(2): 183–200. Publisher Full Text\n\nWilsdon J, Allen L, Belfiore E, et al.: The metric tide: Report of the independent review of the role of metrics in research assessment and management. Higher Education Funding Council for England; 2015. Reference Source\n\nWood F: The cult of the quantifiable: The fetishism of numbers in higher education. Prometheus. 2021; 37(1): 8–26.\n\nZhang L, Sivertsen G: The new research assessment reform in China and its implementation. Scholarly Assessment Reports. 2020; 2(1). Publisher Full Text\n\nZuiker SJ, Piepgrass N, Tefera A, et al.: Advancing knowledge mobilization in colleges of education. Int. J. Educ. Policy Leadersh. 2019; 15(1). Publisher Full Text" }	[ { "id": "125407", "date": "22 Apr 2022", "name": "Jefferson Mainardes", "expertise": [ "Reviewer Expertise Education Policy", "Research Ethics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Education research is still the hardest science: a proposal for improving trustworthiness and usability” discusses a highly relevant issue for the academic field: the emphasis given to metrics for the evaluation of academic performance. The article questions the one-size-fits-all assessment (for all areas) and suggests that academic impact should be analyzed from a wide range of research products, and not just a number of citations.\nA relevant aspect of the article is that, in addition to criticizing, it presents six proposals that could be used by faculties of education to complement conventional indicators. This point is highly relevant since the authors present contributions to minimize the existing problem.\nSome suggestions for corrections are the following:\non page 3 it is indicated that the JCR is generated by Scopus and JIF by the Web of Science. The correct information should be that the JCR is generated by Web of Science. It would be useful if other kinds of impact factor could be indicated (e.g. Scimago Journal Ranking – SJR, JIF, and so on).\n\nThe title of the article refers to the idea that research in education is still the hardest science. Some ideas from David C. Berliner (2002) are used. As it is mentioned in the title, readers could expect that this point would be more explored in the article. What are the authors’ arguments in relation to this assertion? The complexity of educational research issues, educational inequalities in many parts of the world, the different onto-epistemological possibilities of theoretical foundations among other could be explored.\nIn general, this is a relevant article and should be accepted for indexing and wide dissemination, including the translation to other languages.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "142161", "date": "18 Jul 2022", "name": "Daniëlle M. L. Verstegen", "expertise": [ "Reviewer Expertise Instructional design", "PBL", "online/blended learning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an opinion article discussing that the current practice in measuring impact of educational research is inadequate. Although the authors are not the first to make this argument, they present an explicit analysis of why it is inadequate and also harmful. They include some example cases and refer elaborately to the literature.\nOne argument that I would be inclined to add is that our current practice discourages research into topics or settings that need attention, but are less easy to publish.\nThere is some overlap between the sections, but not very much.\nThe authors end with a plea to focus more on trustworthiness and usability. That plea is convincing, but how we can judge trustworthiness and usability remains a bit vague.\nThe manuscript ends with 6 guidelines for improvement. These are concrete, but less nuanced than the text before and more clearly targeting Colleges of education (rather than the whole readership). For me, that decreases the trustworthiness and usability of this manuscript.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-230
https://f1000research.com/articles/11-227/v1	24 Feb 22	{ "type": "Case Report", "title": "Case Report: Purtscher-like retinopathy in a patient with lung adenocarcinoma", "authors": [ "Anis Mahmoud", "Asma Zaghdoudi", "Soumaya Boucharb", "Fatma Abid", "Sameh Mbarek", "Hassan Ibn Hadj Amor", "Sawssen Braiek", "Nadia Keskes Boudawara", "Jalel knani", "Riadh Messaoud", "Asma Zaghdoudi", "Soumaya Boucharb", "Fatma Abid", "Sameh Mbarek", "Sawssen Braiek", "Nadia Keskes Boudawara", "Jalel knani", "Riadh Messaoud" ], "abstract": "This case report describes an unreported case of Purtscher-like retinopathy in a patient with pulmonary adenocarcinoma. A 39-year-old man was hospitalized for exploration of a hemoptysis and bilateral blurry vision. Fundoscopic examination showed multiple areas of retinal whitening in the peripapillary area. A chest computed tomography scan then showed a ground glass opacity in the right upper lobe associated to a hilar lymphadenopathy. A thoracotomy and lung biopsy were performed concluding with the diagnosis of lung adenocarcinoma. The patient was treated with high-dose corticosteroids and received Taxol-Carboplatin chemotherapy with good visual outcomes. The article discusses furthermore the importance of including pulmonary adenocarcinoma to the list of systemic conditions for Purtscher-like retinopathy.", "keywords": [ "Purtscher-Like Retinopathy", "Lung Adenocarcinoma", "corticosteroids" ], "content": "Introduction\n\nPurtscher's retinopathy is a rare condition occurring in the context of trauma, and it was first described by Otmmar Purtscher in 1910.1 Patients with similar retinal findings, such as bilateral cotton wool spots and haemorhages in non-traumatic circumstances are labeled “Purtscher-like” retinopathy.2\n\nWe herein describe a previously unreported association of Purtscher-like retinopathy with lung adenocarcinoma. We aim to highlight the importance of identifying this clinical entity and to provide a credible hypothesis about inherent mechanism of this disease.\n\n\nCase report\n\nA 39-year-old Tunisian, unemployed man with no previous pathological history presented to the emergency department for exploration of recurrent hemoptysis.\n\nA chest computed tomography (CT) scan revealed ground glass opacity in the right upper lobe, associated to a hilar lymphadenopathy measuring 42 mm32 mm28 mm with irregular contours and compressing the esophagus [Figure 1].\n\nBased on these results, the patient was admitted to the pulmonary department for further investigation and appropriate treatment including blood transfusion and hydration. Then, he was referred to the ophthalmology department to examine blurred vision present since the onset of his respiratory symptoms.\n\nOn examination, visual acuity was 20/40 in both eyes. The intraocular pressure was 12 mm Hg in both eyes (normal range, 10-21 mm Hg) and bilateral anterior segment examination showed no abnormalities. No defects in pupillary reflexes or eye movements were noted. Dilated fundus examination showed bilateral multiple areas of retinal whitening in the peripapillary area, resembling cotton wool spots [Figure 2].\n\nSwept source optical coherence tomography scans passing through the cotton wool spots showed focal hyperreflectivity in the inner/middle retinal layers [Figure 3].\n\nThe fluorescein angiography was unremarkable, except for mild obscuration of the retinal vasculature corresponding to the areas of cotton wool spots (red arrows) [Figure 4].\n\nOn the basis of fundoscopic findings showing bilateral cotton wool spots limited to the peripapillary area in a patient with no traumatic background, the diagnosis of Purtscher-like retinopathy was made.\n\nA thoracotomy and right upper lobe biopsy were performed leading to the conclusion and diagnosis of lung adenocarcinoma.\n\nThe patient was treated with high-dose intravenous methylprednisolone (1000 mg daily) for three days, followed by oral corticosteroids at a dose of 1 mg/kg/day with progressive degression over two months and received four courses of 200 mg/m2 Taxol and Carboplatin at AUC of 6 mg/mL/min every 3 weeks. No chemotherapy-related adverse effects were noted. Following this treatment, the patient experienced significant clinical improvement and the hemoptysis resolved. A recent chest CT scan showed no remaining masses.\n\nOn the ophthalmic side, the patient's visual acuity improved to 20/20. On dilated fundus examination [Figure 5], there was significant decrease in the number and size of cotton wool spots in both eyes.\n\n\nDiscussion\n\nPurtscher’s retinopathy is an occlusive microvasculopathy, which was first reported by Otmar Purtscher in 1910, in a patient with blanching whitening and hemorrhages at the posterior pole following severe head trauma.1\n\nPurtscher-like retinopathy includes all retinal lesions similar to the description by Otmar Purtshcer in 1910,1 but occurring in non-traumatic context such as patients with acute pancreatitis, connective tissue disorders and lymphoproliferative diseases.2\n\nBoth Purtscher retinopathy and Purtscher-like retinopathy are considered as extremely rare diseases, with a reported combined incidence rate of 0.24 per million.3\n\nOur patient’s ocular findings were bilateral cotton wool spots limited to the peripapillary area. Clinical course and favorable evolution following chemotherapy, appear to be consistent with Purtscher-like retinopathy complicating lung adenocarcinoma.\n\nThe exact pathogenesis of Purtscher and Purtscher-like retinopathy remains controversial. The theory most supported regarding its mechanism is attributed to an embolic occlusion involving the precapillary arterioles.4\n\nIn malignancies, Purtscher-like retinopathy is considered as paraneoplastic disorder in which interactions of tumor cells with vascular endothelium, blood coagulation agents, and platelets contribute to its pathogenesis.5–7\n\nLung adenocarcinoma is a cause of hypercoagulability,8 and numerous reports highlight its association with retinal vascular thromboembolic events.\n\nRonchetto9 described the case of a patient with branch retinal vein occlusion associated with lung carcinoma, and suggested that the retinal microangiopathic changes may result from the hypercoagulable state seen in such a tumor.\n\nMadabhavi et al10 described a central retinal artery occlusion during the evolution of a pulmonary adenocarcinoma. This incident was attributed to the stimulation of the coagulation activation system by tumoral cells.\n\nTreatment of the acute phase of Purtscher’s retinopathy is based on high-dose intravenous corticosteroids, and visual recovery is variable, being directly related to the extent of non-perfusion areas on retinal angiography.11 The visual outcome in our patient was good, which is consistent with the absence of extensive areas of non-perfusion on retinal angiography.\n\nThis case highlights the importance of ophthalmologic examination in patients with lung adenocarcinoma for suggestive findings of Purtcher-like retinopathy. Nevertheless, this association remains poorly explained and requires further documented cases.\n\n\nConclusion\n\nTo the best of our knowledge, this case report is the first to describe the association between lung adenocarcinoma and Purtscher-like retinopathy and the mechanism of this association remains speculative. Despite this, we suggest that lung adenocarcinoma should be included in the list of systemic conditions of Purtscher retinopathy.\n\n\nConsent\n\nThe patient provided informed written consent for the publication of this case report and associated figures.\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\nPurtscher O: Angiopathia retinae traumatica. Lymphorrhagien des Augengrundes. Albr. Graef’s Arch. Ophtalmol. 1912; 82(2): 347–371. Publisher Full Text\n\nAgrawal A, McKibbin MA: Purtscher’s and Purtcher-like retinopathies: a review. Surv. Ophtalmol. 2006; 51: 129–136. PubMed Abstract \| Publisher Full Text\n\nAgrawal A, McKibbin M: Purtscher's retinopathy: Epidemiology, clinical features and outcome. Br. J. Ophthalmol. 2007; 91(11): 1456–1459. PubMed Abstract \| Publisher Full Text\n\nBehrens-Baumann W, Scheurer G, Schroer H: Pathogenesis of Purtscher’s retinapathy. An experimental study. Graefes Arch. Clin. Exp. Ophthalmol. 1992; 230: 286–291. Publisher Full Text\n\nShah RJ, Choudhry N, Leiderman YI: Purtscher-like retinopathy in association with metastatic pancreatic adenocarcinoma and capecitabine therapy. Retin. Cases Br. Rep. 2013; 7(3): 196–197. PubMed Abstract \| Publisher Full Text\n\nTing DSJ, Smith J, Talks SJ: Purtscher-like retinopathy associated with squamous cell carcinoma of the cervix. Int. Ophthalmol. 2018; 38(1): 389–393. PubMed Abstract \| Publisher Full Text\n\nTabandeh H, Rosenfeld PJ, Alexandrakis G, et al.: Purtscher-like retinopathy associated with pancreatic adenocarcinoma. Am J. Ophthalmol. 1999; 128(5): 650–652. PubMed Abstract \| Publisher Full Text\n\nSyrigos K, Grapsa D, Sangare R, et al.: Prospective assessment of clinical risk factors and biomarkers of hypercoagulability for the identification of patients with lung adenocarcinoma at risk for cancer-associated thrombosis: The Observational ROADMAP-CAT Study. Oncologist. 2018; 23(11): 1372–1381. PubMed Abstract \| Publisher Full Text\n\nRonchetto F: Occlusion of a branch of the central retinal vein as a manifestation of hypercoagulability in a patient with lung cancer. A possible paraneoplastic event. Recenti Prog. Med. 1994; 85(2): 108–112. PubMed Abstract\n\nMadabhavi I, Patel A, Anand A, et al.: Central retinal artery occlusion, an early sign of crizotinib resistance in an alk positive adenocarcinoma of lung: A rare case report. Clin. Respir. J. 2018; 12(2): 806–810. PubMed Abstract \| Publisher Full Text\n\nAtabay C, Kansu T, Nurlu G: Late visual recovery after intravenous methylprednisolone treatment of Purtscher’s retinopathy. Ann. Ophthalmol. 1993; 25: 330–333. PubMed Abstract" }	[ { "id": "125329", "date": "17 Mar 2022", "name": "Ayman G. Elnahry", "expertise": [ "Reviewer Expertise Vitreoretinal 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 report describes a rare case of Purtscher-like retinopathy associated with lung adenocarcinoma. The article is well written and well illustrated and adds to the existing body of literature on associations with Purtscher-like retinopathy. It is interesting to elaborate on the possible association between increased intrathoracic pressure with the coughing associated with hemoptysis/valsulva (if it was excessive in this case) in relation to Purtscher-like retinopathy since chest trauma is a known cause of Purtscher retinopathy which may occur through increased intrathoracic pressure following the chest trauma leading to impaired venous drainage from the eye and stasis with resultant hemorrhage and CWS.\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": "125328", "date": "22 Mar 2022", "name": "Meriem Ouederni", "expertise": [ "Reviewer Expertise medical retina", "cornea", "cataract" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes an interesting unreported case of Purtscher-like retinopathy in a patient with pulmonary adenocarcinoma.\nComplete illustration of the case was provided. The case adds a new systemic condition associated with Putscher-like retinopathy.\nIn the discussion section, pathogenesis was discussed even if the association remained poorly explained. Further documented cases are needed for better understanding of the relationship.\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": "125326", "date": "22 Mar 2022", "name": "Najeh Hcini", "expertise": [ "Reviewer Expertise infectious diseases - immunity -Virus" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 an interesting case report of a young person who developed Purtscher-like retinopathy in association with lung adenocarcinoma.\nThe authors also explained well the possible pathophysiology of this entity as well as the risk of retinal vascular thrombosis associated with lung adenocarcinoma.\nThe discussion section is well written and organised, which is sure to attract readers.\nConclusion:\n\n\"To the best of our knowledge\": please remove this phrase. \"Despite this\": please rephrase the last sentence.\nIn total: Overall, I found this case report interesting and approve it for indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-227
https://f1000research.com/articles/11-226/v1	24 Feb 22	{ "type": "Genome Note", "title": "The complete mitogenome sequence of clam (Corbicula fluminea)", "authors": [ "Qing Luo", "Jie Pi", "Yangxin Tang", "Cong Zeng", "Deliang Li", "Qing Luo", "Jie Pi", "Yangxin Tang", "Cong Zeng" ], "abstract": "The global invasion of the genus Corbicula has caused serious ecological and economic problems. The species of Corbicula fluminea stands out amongst the greater part of freshwater invaders around the world. Here, we sequenced the entire mitogenome of the Corbicula fluminea, which were gathered from Dongting Lake, in the Hunan province of central China. The circular genome is 17,073 bp long. The raw reads were obtained from the platform of Illumina HiSeq 2500, and assembled by the MITObim method followed by alignments to related species. The entire dataset was deposited at the NCBI Short Read Archive via accession number SRR14692229 and NCBI GenBank with accession number MZ231034.1.", "keywords": [ "Corbicula fluminea", "mitochondrial genome", "phylogenetic analysis" ], "content": "Introduction\n\nClams of the genus Corbicula are currently spread worldwide, and they cause great ecological threats and tremendous impacts in the ecological system (Counts 1981; Gomes et al. 2016; Peñarrubia et al. 2017; Hünicken et al. 2019; Douglass et al. 2020). However, their taxonomic status and systematic status are still unclear (Wang et al. 2018; Haponski 2019; Bodon et al. 2020; Ramli et al. 2020). Corbicula fluminea is an important component of Asian freshwater macrobiota and has always been consumed as food in East Asia (Wang et al. 2014; López-Soriano et al. 2018; Zhang et al. 2019; Sano et al. 2020). In this work, the complete mitochondrial genome of Corbicula fluminea from Dongting Lake was sequenced, and the phylogenetic relationships among Venerida were investigated. These results could contribute to distinguishing the taxonomic placement and systematic status of genus Corbicula in further studies.\n\n\nMethods\n\nThe specimens (Corbicula fluminea) used in this work was collected from Dongting Lake (geographic location: 28°46′45″N, 112°41′22″E), in Hunan province, China in August 2018.\n\nAfter sampling, the living specimens were stored in College of Animal Science and Technology, Hunan Agricultural University, the voucher number is DT1808-118.\n\nTo obtain the total genomic DNA, the 40mg frozen muscle of the foot was derived from the specimen. DNA was obtained by DNeasy™ Blood & Tissue extraction kit (Qiagen, Hilden, Germany). The library was conducted by Berry Genomics Co. Ltd (Beijing, China) according to the Illumina TruSeq Nano DNA library Prep Kit and the manufacturer’s recommendations. DNA fragments were selectively enriched using Illumina PCR Primer (F: 5′-AATGATACGGCGACCACCGAGA-3′ and R: 5′-CAAGCAGAAGACGGCATACGAGT-3′) Cocktail in a PCR reaction with the following cycling conditions: 95°C for 3 minutes, followed by 8 cycles of 98°C for 20 seconds, 60°C for 15 seconds and 72°C for 30 seconds, with a final extension step at 72°C for 5 minutes. The raw reads of the mitogenome sequence were obtained from the platform of Illumina HiSeq 2500. The adaptors and low sequencing qualities reads (N bases exceeding 10% and more than 50% of phred quality score ≤ 5) were trimmed and reduced using the program Trimmomatic v 0.38.0 (Bolger et al. 2014).\n\nThe sequence reads of untrimmed high-quality were assembled by the program MITObim (Hahn et al. 2013). The annotation was performed using MITOS WebServer (Bernt et al. 2013) for the entire mitogenome of Corbicula fluminea from Dongting Lake, and adjusted manually in Geneious Prime v2020.2.2 based on the published mitogenomes of Venerida species.\n\nTo validate our data, the CDS sequences of 19 Venerida species (GenBank accession numbers are shown in Figure 2) were aligned. The phylogenetic analysis among Venerida was investigated using maximum-likelihood (ML) and Bayesian inference (BI) approaches. The ML tree was estimated by RAxML version 8.2.12 (Stamatakis 2014) and the BI tree was estimated by MrBayes version 3.2.7 (Ronquist et al. 2012), with GTR-GAMMA model. RAxML was analyzed with 1,000 bootstrap replicates (-m GTRGAMMAI -f a -x 1 -N 1 -p 1 -N 1000), 1 million generations Markov chain Monte Carlo iterations (nCat=4) were analysed and every 1,000 generations were sampled with the initial 10% of samples removed as “burn-in” in MrBayes. The average standard deviation of split frequencies (<0.01) was used to assess the convergence.\n\nA phylogenetic relationship was estimated based on 13 protein-coding genes sequences from the mitogenome sequences of Corbicula fluminea from Dongting Lake and other 19 Venerida species (Figure 2). The study has provided a complete mitochondrial genome of Corbicula fluminea from Dongting Lake. As a phylogenetic tool, it could contribute to systematic and comparative analysis for resolving evolutionary relationships.\n\nThe nodal numbers indicate the bootstrap support values (left) and the posterior probability (right). Genbank accession numbers for the sequences are indicated next to the species names.\n\n\nResults\n\nThe complete circular mitogenome of Corbicula fluminea from Dongting Lake (GenBank accession no. MZ231034.1) is 17,073 bp in total length. As other Venerida mitogenomes, 37 mitochondrial structural genes were found in the mitogenome of Corbicula fluminea from Dongting Lake, including 13 protein-coding genes, two ribosomal RNA genes (12S and 16S rRNA) and 22 transfer RNA genes (Figure 1). Both are encoded on the positive strand.\n\n\nData Availability\n\nNCBI Gene: Complete mitochondrial genome of Corbicula sp. DT118, SRA\n\nAccession number: SRR14692229\n\nAccession number URL: https://www.ncbi.nlm.nih.gov/sra/SRR14692229\n\nNCBI Gene: Corbicula sp. QL-2021 mitochondrion, complete genome\n\nAccession number: MZ231034.1\n\nAccession number URL: https://www.ncbi.nlm.nih.gov/nuccore/MZ231034.1\n\n\nEthical approval\n\nAll the clams and experimental protocols involving experiments of this study were approved by the Animal Ethics Committee of Hunan Agricultural University, Hunan, China.", "appendix": "References\n\nBernt M, Donath A, Jühling F, et al.: MITOS: improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 2013; 69(2): 313–319. PubMed Abstract \| Publisher Full Text\n\nBodon M, López-Soriano J, Quiñonero-Salgado S, et al.: Unraveling the complexity of Corbicula clams invasion in Italy (Bivalvia: Cyrenidae). Bollettino Malacologico. 2020; 56: 127–171.\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. PubMed Abstract \| Publisher Full Text\n\nCounts CL: Corbicula fluminea (Bivalvia: Sphaeriacea) in British Columbia. Nautilus. 1981; 95: 12–13.\n\nDouglass S, Reasor E, Tiemann J, et al.: Recent evaluation of Corbicula form D distribution in the Midwest, USA. Am. Midl. Nat. 2020; 183(1): 136–142.\n\nHahn C, Bachmann L, Chevreux B: Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach. Nucleic Acids Res. 2013; 41(13): e129–e129. PubMed Abstract \| Publisher Full Text\n\nHaponski AE, Foighil DÓ: Phylogenomic analyses confirm a novel invasive North American Corbicula (Bivalvia: Cyrenidae) lineage. PeerJ. 2019; 7: e7484. PubMed Abstract \| Publisher Full Text\n\nHünicken LA, Abrameto M, Bonel N: Corbicula at its southernmost invasion front in Patagonia: unusual low density and asymmetric trait responses to varying environmental conditions. J. Molluscan Stud. 2019; 85(1): 143–153. Publisher Full Text\n\nLópez-Soriano J, Quiñonero-Salgado S, Cappelletti C, et al.: Unraveling the complexity of Corbicula clams invasion in Lake Garda (Italy). Adv. Oceanogr. Limnol. 2018; 9(2) Publisher Full Text\n\nGomes C, Sousa R, Mendes T, et al.: Low genetic diversity and high invasion success of Corbicula fluminea (Bivalvia, Corbiculidae) (Müller, 1774) in Portugal. PLoS One. 2016; 11(7): e0158108. PubMed Abstract \| Publisher Full Text\n\nPeñarrubia L, Araguas RM, Vidal O, et al.: Genetic characterization of the Asian clam species complex (Corbicula) invasion in the Iberian Peninsula. Hydrobiologia. 2017; 784(1): 349–365. Publisher Full Text\n\nRamli MZ, Ayyapan V, Yusoff A, et al.: Phenotype and Genotype Characterisation of the Asian Clam of the Genus Corbicula Megerle von Mϋhlfeld, 1811 (Venerida, Cyrenidae) from the East Coast of Peninsular Malaysia. Borneo Journal of Resource Science and Technology. 2020; 10(1): 24–36. Publisher Full Text\n\nRonquist F, Teslenko M, van der Mark P , et al.: MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012; 61: 539–542. PubMed Abstract \| Publisher Full Text\n\nSano N, Houki S, Kodan A, et al.: Genetic confirmation of “egg parasitism” in androgenetic freshwater Corbicula clams by paternity testing using microsatellite DNA markers. Plankton and Benthos Research. 2020; 15(1): 58–62. Publisher Full Text\n\nStamatakis A: RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–1313. PubMed Abstract \| Publisher Full Text\n\nWang GP, Zhang T, Zhang J, et al.: Morphological and molecular differentiation of genus Corbicula suggests that two species are sympatrically distributed in Datong Lake in the Central Yangtze River Basin. Zool. Stud. 2014; 53(1): 1–8. Publisher Full Text\n\nWang JP, Li DL, Ze C, et al.: Genetic diversity and reproductive characteristics of Corbicula fluminea from the Laodao River in Liuyang city. Acta Hydrobiologica Sinica. 2018; 42(5): 913–918.\n\nZhang T, Yin J, Tang S, et al.: The complete mitogenome of clam Corbicula fluminea determined using next-generation and PacBio sequencing. Mitochondrial DNA Part B. 2019; 4(1): 1660–1661. Publisher Full Text" }	[ { "id": "125320", "date": "24 Mar 2022", "name": "Xiaotong Wang", "expertise": [ "Reviewer Expertise Mollusc physiology and molecular biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the assembly and annotation of a clam mitogenome. It’s a brief note that provides some useful background about the species, Corbicula fluminea, and these data can contribute to distinguish the taxonomy and systematic status on the genus Corbicula in further studies.\nThe details of the analysis and the data is clear. I have 2 minor revisions:\nInclude version number of MITObim and MITOS.\n\nThe name of the species should be unified in the manuscript and the NCBI GenBank.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] }, { "id": "140845", "date": "18 Jul 2022", "name": "Arsalan Emami-Khoyi", "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\nIn my opinion, the paper contributes significantly to the knowledge base of this species. No major flaws in the analysis were noticed.\nThe authors need to better justify why this particular clam has been chosen for sequencing.\nThe authors need to clarify in which mode Mitobim has been used. Has a conserved template sequence from a closely related species been used as a starting seed?\n\na) The title needs to be changed to \"Asian\" clam (Corbicula fluminea).\nb) Please note that ribosomal RNA are not genes.\nc) Great \"ecological threats\" and \"tremendous impacts\" need to be clarified.\nd) How were the Bayesian runs trace files evaluated for convergence? Why was the Effective Sample Size not reported?\n\nAre the rationale for sequencing the genome and the species significance clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-226
https://f1000research.com/articles/11-224/v1	24 Feb 22	{ "type": "Research Article", "title": "Association between S-COMT activity and impulsive and premeditated aggression in a population of violent offenders: preliminary results of a cross sectional study", "authors": [ "Jacinto Azevedo", "Cláudia Carvalho", "Maria Paula Serrão", "Rui Coelho", "Maria Augusta Vieira-Coelho", "Margarida Figueiredo-Braga", "Cláudia Carvalho", "Maria Paula Serrão", "Rui Coelho", "Maria Augusta Vieira-Coelho", "Margarida Figueiredo-Braga" ], "abstract": "Aggression can be conceptualised as a physical act towards another person, verbal offenses, destructive acts towards objects, and self-inflicted harmful acts. It is highly frequent in the context of Antisocial Personality Disorder (ASPD) and has been correlated to disturbances in the dopaminergic system. In the prefrontal cortex, the dopamine metabolism depends on catechol-O-methyltransferase (COMT). DRD2 receptors also play a role in the expression of aggression by modulating dopamine metabolism, in the striatum. In this study, we evaluated the association between COMT activity and type of aggression, in a sample of violent male offenders. Participants were subjected to sociodemographic, clinical, and psychometric evaluation through standardised instruments. Erythrocyte S-COMT activity was measured, and COMT and the DRD2 genotypes were analysed. Individuals displaying impulsive aggression showed lower S-COMT erythrocyte activity (p=0.026) and lower frequency of Val/Val (rs4680) genotype than individuals with premeditated aggression (p=0.047). S-COMT erythrocyte activity was positively correlated with the PCL-R total score (r=0.34; p=0.018). In conclusion, our preliminary results indicate that COMT can be associated to different aggression types in violent offenders, and it can represent a possible pharmacological target for the treatment of impulsive and premeditated aggression, in incarcerated patients.", "keywords": [ "Aggression", "COMT", "Antisocial personality disorder", "Impulsivity", "BIS-11" ], "content": "Introduction\n\nAggression can manifest itself through physical acts directed against other individuals, verbal offenses, destructive acts towards objects, and self-inflicted harmful acts (Siever, 2008). In this study, we limited aggression to physical aggression directed against another individual. An act of aggression can be categorized as impulsive or premeditated, with distinct manifestation over time; although there may be a predominant type (Meloy, 2006).\n\nAggression is part of several psychiatric syndromes and is particularly common in antisocial personality disorder (ASPD) (American Psychiatric Association, 2013), in which physical aggression is one of the core symptoms (Bourne, 2010). Aggressive behaviour conducts often to crime, with subsequent episodes of imprisonment (Pingault et al., 2013). Nevertheless, even during periods of incarceration, aggressive behaviour is maintained (Morana, Arboleda-Flórez, & Câmara, 2005; Wolff, Blitz, Shi, Bachman, & Siegel, 2006). Some of these individuals express aggression since early childhood and pharmacotherapy has shown reduced efficacy (Felthous, Lake, Rundle, & Stanford, 2013). Overall, the individuals remain unable to reduce this behaviour through common learning and education. This reinforces the need for an effective assessment of the displayed aggression type, as well as for further research of biological targets for pharmacological intervention (Pingault et al., 2013).\n\nThe most relevant predictors for aggression are criminal history, hostile behaviour, impulsivity, antisocial personality, recent drug or alcohol misuse, ‘positive symptoms’ of psychosis, and non-adherence to therapy (Farrington, 2011; Witt, van Dorn, & Fazel, 2013).\n\nThe relevance of the genetic study of human aggression is highlighted by the fact that aggression represents a behaviour that the human species has upheld over time, suggesting its evolutionary value (Wrangham, 2018). The heritability of aggressive behaviour can reach 50%, and genetic factors are related to the aetiology of aggression, in particular that starting in childhood (Blonigen & Krueger, 2005; Laucht, Brandeis, & Zohsel, 2013).\n\nIn a recent study, we have confirmed that, although impulsive aggression was predominant in a population of male ASPD offenders, those who showed higher scores of psychopathic traits were less prone to exhibit impulsive aggression, and tended to display premeditated aggression (J. Azevedo, Vieira-Coelho, Castelo-Branco, Coelho, & Figueiredo-Braga, 2020). An individual showing premeditated aggression can have a higher risk for aggression recidivism: in these cases, the use of pharmacological interventions has no positive results (Swogger, Walsh, Christie, Priddy, & Conner, 2015). Hence, it is mandatory to characterise the aggression type in offenders and find the biological variants that could be therapeutic targets.\n\nThe neurobiology of aggression in healthy and psychiatric populations has been associated with changes in the dopamine metabolism (Panasiuk, Hertz, & Gale-Grant, 2019; Rosell & Siever, 2015). The dopaminergic system function has been linked to angry-impulsive personality traits (Joyce et al., 2009) and to a detached personality (Jönsson et al., 2003). In humans, pharmacological agents which increase dopamine levels can lead to aggressive behaviour and impulse control disorders (Azevedo J, Esteves M, Rosas MJ, 2010; Canário et al., 2019).\n\nThe dopamine metabolism in the prefrontal cortex (PFC) and limbic system depends mostly on the Catechol-O-methyltransferase (COMT) activity. The enzymatic activity of COMT, including S-COMT, has been associated with physical aggression in several studies, opening several possibilities for future research on the utility of this enzyme as a target for the treatment of several dysfunctions (Craig & Halton, 2009; Volavka et al., 1997).\n\nThe most analysed COMT polymorphism is the COMT Val158Met (rs4680) that is distributed in Caucasian populations as follows: Met/Met 28%, Met/Val 51%, Val/Val 21% (González-Castro et al., 2013). This polymorphism (responsible for a decrease in COMT activity) has been associated with aggression in different psychiatric populations. For example, the Met/Met genotype (lower enzymatic activity of COMT) has been associated with aggression in schizophrenia patients (Han, Park, Na, Kee, & Lee, 2004). Conversely, the Val/Val genotype (higher enzymatic activity of COMT) is associated to aggression in samples of patients with attention deficit hyperactivity disorder (ADHD) and extreme antisocial behaviour (Langley, Heron, O’Donovan, Owen, & Thapar, 2010). These contradictory results may be due to the lack of clinical characterization and definition of the type of aggression, in the studied samples, and to the possible variation of frequency of the distribution of this COMT polymorphism, in different populations (Reidy et al., 2017). The role of COMT activity in decision-making processes, through the modelling of the availability of dopamine in the PFC, could predict the type of aggression.\n\nIt has been proposed that antisocial individuals, having the Met/Met genotype, may present stable cognitive performance and preserved emotional functioning, but lower resistance to emotional stress, being more sensitive to social rejection and to the need of reward (Yildirim & Derksen, 2015). On the other hand, it has also been proposed that antisocial individuals with the Val/Val genotype would show reduced cognitive performance, emotional insensitivity, higher resistance to emotional stress, and insensitivity to social rejection and reward (Yildirim & Derksen, 2015). These theoretical proposals may suggest that antisocial individuals with the Val/Val genotype may display more psychopathic personality traits related to emotional insensitivity measured by the facets interpersonal (F1) and affective (F2) of the Psychopathic Checklist-Revised (PCL-R) (Hare, 2003). These personality traits have been consistently pointed out as predictors of premeditated aggression in individuals with extreme antisocial behaviour (J. Azevedo et al., 2020).\n\nThe COMT Val158Met polymorphism determines the dopamine concentration in the PFC, but not in the striatum (Hirvonen et al., 2010). In fact, we can find only a few dopamine D2 receptors in the PFC (MISSALE, NASH, ROBINSON, JABER, & CARON, 1998). However, they are abundant in the striatum, meaning that, to evaluate the dopaminergic component of aggression we should also analyse dopamine receptors. The A1 (T) allele of dopamine D2 receptor/ankyrin repeat and kinase domain containing 1 (DRD2/ANKK1) TaqIA (rs1800497) single nucleotide polymorphism has been associated with reduced striatal D2/D3 receptor availability. This genetic variant has been referred to contribute to significant individual differences in human striatal function, neuropsychiatric disease risk, and pharmacological response (Eisenstein et al., 2016). It has been associated with addiction, impulsivity and antisocial personality traits (Ponce et al., 2008; Rivera-Iñiguez, Panduro, Ramos-Lopez, Villaseñor-Bayardo, & Roman, 2019). In addition, it may also play a role in aggression due to its influence on psychopathy phenotypes (Ponce et al., 2008).\n\nImpulsive and premeditated aggression may be distinguished by biological, psychological, psychosocial, and clinical variables (Meyer, Cummings, Proctor, & Stahl, 2016). The dopaminergic system appears to be a relevant player in this distinction, informing the risk for aggression recidivism and the effectiveness of pharmacological interventions (Swogger et al., 2015).\n\nIn this study, we aimed to evaluate the dopaminergic function in imprisoned violent offenders, through the assessment of the correlation between COMT activity and aggression type. For this, we used a cross sectional approach in a sample of violent male offenders and confirmed the results by genotyping.\n\n\nMethods\n\nThis study was conducted in a medium/high security penal institution for male offenders, in the North of Portugal. The sample included 46 male inmates that were referred to the Psychiatry clinical services, after preforming acts of physical aggression towards other inmates. The research protocol was approved by an Ethics Committee from Centro Hospitalar e Universitário de São João (Document number 48.14) and by the hosting institution, the General Directorate for Probation and Prison Services.\n\nSubjects were recruited through the use of a convenience sampling strategy between January and March of 2015. The primary requirement for inclusion in the study was the evidence of acts of physical aggressions towards other inmates. Participants should also be over 18 years old. The ability to read and to provide their written informed consent was also taken into consideration. Participants were excluded if the aggression occurred in the context of an acute neurological disorder (e.g., epilepsy, confusional states) or as an acute episode of a major psychiatry disorder (e.g., non-organic psychosis, major depression).\n\nIn accordance with the Declaration of Helsinki, informed consent was obtained from the legal guardian of the subjects, after being elucidated about the procedures.\n\nThe participants that agreed to participate were interviewed and went through a blood sample collection, in the clinical department of the penitentiary institution.\n\nIndividuals were diagnosed by a forensic psychiatrist (JA) using a standardised interview – the Mini-International Neuropsychiatric Interview (MINI). Participants were assessed by means of the Portuguese version (Amorim, Lecrubier, Weiller, Hergueta, & Sheehan, 1998; Lecrubier et al., 1997). The psychometric assessment of participants included a battery of standardised instruments: the Psychopathy Checklist-Revised (PCL-R), the Barratt Impulsivity Scale Version 11 (BIS-11), and the Impulsive/Premeditated Aggression Scale (IPAS).\n\nWe opted for a cross sectional approach that allowed us to compare, at a specific point in time, two groups within our population: group P (individuals with premeditated aggression) and group I (individuals with impulsive aggression).\n\nThe biological variables of this study were S-COMT erythrocyte activity, and COMT Val158Met (rs4680) and DRD2/ANKK1 TaqIA (rs1800497) polymorphisms.\n\nThe PCL-R measures psychopathic traits by collecting information from clinical records and applying a semi-structured interview (Hare, 2003). The 20 items that compose the PCL-R are scored as absent (0), present to some degree (1), or fully present (2), providing a maximum total score of 40 points. The PCL-R is a four-factor model comprising interpersonal, affective, lifestyle, and antisocial facets. The structural properties of PCL-R were previously validated in Portuguese samples (Gonçalves, 1999).\n\nThe BIS-11 is a self-report questionnaire used for assessing general impulsivity (Patton, Stanford, & Barratt, 1995). The current scale version contains 30 items that are rated from 1 (rarely/never) to 4 (almost always/always). Factor analyses reveal six first-order factors (Attention, Cognitive Instability, Motor, Perseverance, Self-Control, and Cognitive Complexity), and three second-order factors (Attentional, Motor, and Non-planning). The structural properties of BIS-11 were replicated in Portuguese-speaking subjects (von Diemen, Szobot, Kessler, & Pechansky, 2007).\n\nIPAS is a 30-item self-report questionnaire used to rate aggressive acts that occurred over the previous six months (Stanford et al., 2003). In details, participants were asked to complete the IPAS questionnaire considering their aggressive acts in the last 6 months. The questionnaire comprises fifteen items focused on impulsive aggressive (IA) characteristics and 15 items focused on premeditated aggressive (PM) characteristics. Items are scored on a five-point Likert scale ranging from 1 (strongly disagree) to 5 (strongly agree).\n\nIA and PM can be scored either dimensionally or categorically. To characterize aggressive behavior as predominately impulsive or premeditated, items for which the individual answered “strongly agree” or “agree” were rated as positive and the total number of positive items was then determined for both IA and PM scales. The aggressive behavior of those individuals showing a higher percentage of positive IA characteristics was characterized as predominately impulsive, whereas those showing a higher percentage of positive PM characteristics were characterized as predominately premeditated. Individuals showing the same percentage of positive IA and PM characteristics were not characterized.\n\nThe structural properties of IPAS were previously validated in the Portuguese forensic and general population (J. C. Azevedo, Pais-Ribeiro, Coelho, & Figueiredo-Braga, 2018; Cruz et al., n.d.).\n\nErythrocyte S-COMT was obtained from washed erythrocytes submitted to haemolysis, as previously described (Amorim-Barbosa, Serrão, Brandão, & Vieira-Coelho, 2016). Venous blood samples were collected between 08.00 and 09.00 a.m., after an overnight fast, and kept on ice in K3EDTA tubes, until processing. Blood samples were centrifuged at 1500 g, for 10 minutes at 4 °C; the plasma was removed and the uppermost cell layer was separated for genetic analysis. Afterwards, a volume of cold 0.9% NaCl solution was added to the erythrocytes and the mixture was gently vortexed. Thereafter, the tubes were centrifuged (at 1500 g, 10 minutes, 4 °C), and the supernatant was discarded. This process was repeated twice. Washed erythrocytes were stored at -70 °C, until the enzyme assay was carried out. On the day of the experiment, the frozen erythrocytes were thawed on ice.\n\nHaemolysis was conducted at a ratio of 4:1 (water:erythrocytes; V:V). Following vigorous mixing, the tubes stood on ice for 10 minutes. Then, the tubes were centrifuged at 20,000 g, for 20 minutes at 4 °C, and the supernatant was collected for the assay of erythrocyte S-COMT. The protein content was determined using human serum albumin as standard (Bradford, 1976).\n\nCOMT activity was determined by the ability of enzyme preparations to methylate adrenaline to metanephrine. In the kinetic studies, COMT was incubated with concentrations of adrenaline of 3, 10, 30, 100, 300, 1000, and 3000 μmol L-1. The reaction was stopped with perchloric acid. The samples were kept at 4 °C for two hours, and then centrifuged (5400 g, 10 minutes, 4 °C). 500 ml aliquots of the supernatant, filtered on 0.22 mm pore size Spin-X filter tubes (Costar), were used for the assay of metanephrine.\n\nAliquots of samples from the COMT assay were assayed for metanephrine by HPLC with electrochemical detection, as described by Vieira-Coelho (Vieira-Coelho & Soares-da-Silva, 1999). In brief, aliquots of 20 or 50 mL were injected into the chromatograph. The chromatographic system consisted of a pump (Gilson 307) and a stainless steel 5 mm ODS2 column (Biophase; Bioanalytical Systems, West Lafayette, IN, USA), of 25 cm length and 4.6 mm diameter; samples were injected by means of an automatic sample injector (Gilson 231) connected to a Gilson dilutor (Gilson 401). The mobile phase was a degassed solution of citric acid 0.1 mM, sodium octylsulphate 0.5 mM, sodium acetate 0.1 M, Na2EDTA 0.17 mM, dibutylamine 1 mM, and methanol (10 % v/v), adjusted to pH 3.5 with PCA 2 M and pumped at a rate of 1.0 mL/min. The detection was carried out electrochemically with a glassy carbon electrode, an Ag/AgCl reference electrode, and an amperometric detector (Gilson 142); the detector cell was operated at 0.75 V. The current produced was monitored using the Gilson Unipoint HPLC software. The lower limit of detection of metanephrine ranges from 350 to 1000 fmol.\n\nThe most common kind of enzyme kinetics experiment is to vary the concentration of substrate and measure enzyme velocity. The goal is to determine the enzyme's KM and Vmax according to the Michaelis-Menten model (Lorsch, 2014):\n\nIn which, Vmax is the maximum enzyme velocity and represents the velocity of the enzyme extrapolated to very high concentrations of substrate, KM is the Michaelis-Menten constant and represents the substrate concentration needed to achieve a half-maximum enzyme velocity and S is the substrate concentration.\n\nS-adenosyl-L-methionine, DL-metanephrine and adrenaline (bitartrate salt) were purchased from Sigma Chemical Co. (St Louis, MO).\n\nGenomic DNA extraction from leukocytes was performed following the manufacturer’s instructions of the Quick-DNA Plus Kits (Zymoresearch, CA, USA). A 100 ng/μL DNA aliquot was stored at -80 °C until use.\n\nAllelic discrimination for the COMT Val158Met (rs4680) and DRD2/ANKK1 TaqIA (rs1800497) polymorphisms were determined through real-time polymerase chain reaction (PCR) technique, using a TaqMan SNP genotyping assay with fluorogenic probes (Applied Biosystems, Foster City, CA). Briefly, 15 ng of DNA was amplified in a total volume of 8 μL containing 0.2 μL of a minor groove binder (MGB) probe solution (Applied Biosystems) and 4 μL of TaqMan universal polymerase chain reaction master mix (Applied Biosystems). PCR conditions were provided by the manufacturer: 40 cycles of 95°C denaturation (15 sec), 60°C anneal/extension (1 min).\n\nThermal cycling and fluorescence signal genotyping were performed through the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). A positive control for each possible genotype and a negative control were included in each 96-well plate.\n\nData were summarised using descriptive statistics: continuous variables are presented as mean (standard deviation) or median (interquartile range [Q1, Q3]), while categorical variables are presented as absolute or relative frequencies. Continuous variables were subjected to normality testing (Kolmogorov–Smirnov test) and, when the normality assumption was validated, Student T test, and Chi-square test were used to compare data in premeditated and impulsive aggression groups. When data did not meet the requirements of parametric tests, non-parametric Mann–Whitney was used. A p value of <0.05 was considered to be statistically significant (J. Azevedo et al., 2020).\n\nThe degree of association between variables with normal distribution (COMT and PCL-R) was measured by the Spearman correlation coefficient.\n\nA logistic regression analysis was employed to investigate the significant variables associated with the prediction of aggression categorisation using Vittinghoff’s recommendations for small samples (Vittinghoff, Eric, McCulloch, Charles E, Glidden, David V, Shiboski, 2007). Using a forward, stepwise, conditional methodology, ASPD, PCL-R total score, facet 1 (interpersonal) and facet 2 (affective), S-COMT erythrocyte activity and COMT genotype were included in the analysis.\n\nMichaelis–Menten constant (Km) and maximum velocity (Vmax) values for COMT activity were calculated by nonlinear regression analysis using the GraphPad Prism statistics software package, version 6.0 for Windows (GraphPad Software, La Jolla, CA, USA) (Amorim-Barbosa et al., 2016). Statistical analysis was performed by one-way analysis of variance ANOVA using Newman–Keuls multiple comparison test to compare values (p<0.001).\n\nAnalyses were carried out using IBM SPSS Statistics for Mac, Version 26.0 (Armonk, NY, USA: IBM Corp.).\n\n\nResults\n\nThe sample comprised 46 offenders with a median age of 35 (Q1: 31, Q3: 41.5) years, of which 33 were single (72%), and 29 had children (63%). The median education level was 7.0 (Q1: 6.0; Q3: 9.5) years. At the time of assessment, the median length of imprisonment was 114.0 (Q1: 60.9; Q3: 175.5) months. Besides having committed aggressive acts towards other inmates, a total of 25 individuals (54%) had also a history of being convicted for violent crimes, such as physical assault, murder, or attempted murder.\n\nConsidering clinical psychiatric disorders in the entire sample, 34 offenders received the diagnosis of antisocial personality disorder (71%), 20 showed psychopathy (44%), 26 presented SUD (57%), 10 had depressive disorders (21%), and 14 displayed anxiety disorder (30%).\n\nResults from aggression characterization (IPAS) showed that impulsive aggression was detected in 78.2 % of the participants (n=36), whereas the premeditated type was detected in 21.8% (n=10). Thus, offenders were allocated in two different groups according with the aggression type: Group I – individuals with impulsive aggression, and Group P – individuals with premeditated aggression.\n\nBoth groups presented similar sociodemographic and criminal characteristics, as well as prevalence of substance use, depressive and anxiety disorders. In addition, both groups presented similar total BIS-11 scores, showing comparable values of attentional, motor, and non-planning dimensions of impulsivity (Table 1).\n\n1 : n (%).\n\n2 : mean ± SD.\n\n3 : median (Q1; Q3).\n\na : Chi-square Test.\n\nb : Student T test.\n\nc : Mann-Whitney Test.\n\nIn contrast, we found that offenders with premeditated type of aggression presented higher prevalence of ASPD (p=0.004), and a higher score for psychopathic traits given by total PCL-R (p=0.001), facet 1 (p=0.011) and 2 (p=0.014) scores, when comparing to impulsive aggression group (Table 1).\n\nErythrocyte S-COMT activity (pmol/mg prt/h) was significantly higher (p<0.05) in offenders with premeditated aggression (22±5) than in those of the impulsive aggression group (18±3). These results were obtained with a single concentration of substrate (adrenaline, 1000 μM), and although significant, the difference between groups was small and its impact was difficult to evaluate. Thus, we decided to characterise enzyme kinetics in 6 individuals from each group. In these experiments, increasing concentrations of adrenaline (1, 3, 10, 30, 100, 300, 1000 and 3000 μM) were used as substrate. Substrate versus velocity curves for Erythrocyte S-COMT activity are shown in Figure 1. Kinetic analyses revealed that the maximum enzyme velocity (Vmax) value was significantly higher (p=0.0001) in offenders with premeditated aggression than in those with impulsive aggression (40.1±2.4 vs 21.2±1.2 pmol/mg prt/h). The Km value (in μM) was similar in both groups (896±141 vs 874±136).\n\nIn addition, S-COMT activity showed to be a predictor of the BIS-11 score (R2=0.12; p=0.022).\n\nConsidering all the sample (n=46), we found that S-COMT erythrocyte activity was positively correlated to PCL-R total score (Pearson correlation=0.34; p=0.018), (Figure 2). However, we could not find any predictor of PCL-R.\n\nThe sample comprised 4 individuals (8.7%) with the Met/Met genotype, 26 (56.5%) with the Met/Val, and 16 (34.8%) with the Val/Val genotype. The results regarding genotype were in agreement with the results of functional enzyme activity, since the S-COMT erythrocyte activity was lower in the Met/Met genotype individuals than in those with Met/Val, and also lower than those with Val/Val genotype (p=0.0001) (Figure 3).\n\nFurthermore, offenders with premeditated aggression had a higher prevalence of the Val/Val genotype than individuals with impulsive aggression (p=0.047).\n\nRegarding the DRD2/ANKK1 TaqIA polymorphism, the sample comprised 13 (28.3%) individuals with A1 allele, with similar distribution in the impulsive and premeditated groups (22.0% vs 29.7%; p=0.07).\n\nThe premeditated and impulsive groups showed differences in ASPD, PCL-R total score, facet 1 (interpersonal) and facet 2 (affective), S-COMT erythrocyte activity and COMT genotype. As so, these variables were included in a logistic regression model performed with the objective of identifying potential predictive factors.\n\nPCL-R reliably distinguished between impulsive and premeditated aggression (Chi-square=16.7; p=0.001; df =1; Nagelkerke’s R2=0.55). The exponential beta value for PCL-R was 1.44 (CI=1.12, 1.85), meaning that, for each increase in one unit of the PCL-R, the OD for premeditated aggression increased by 1.44.\n\n\nDiscussion\n\nIn this preliminary study, we have found that male offenders displaying impulsive aggression showed lower S-COMT erythrocyte activity than individuals with premeditated aggression. Due to the lack of previous evidence on this subject, we opted to further validate the obtained results by adding genetic studies to our initial investigation plan. The results evidenced that impulsive and premeditated aggression were associated with different genotypes of the COMT Val158Met polymorphism and with S-COMT erythrocyte activity. In detail, individuals with premeditated aggression had a higher prevalence of the Val/Val genotype and showed higher values of erythrocyte S-COMT activity. In theory, these individuals may have lower DA availability in PFC.\n\nThe evaluation of S-COMT activity followed by COMT Val158Met polymorphism studies confirmed that functional (enzyme activity) and genetic results were concordant. In fact, the activity of erythrocyte S-COMT was different depending on the genotype: higher in the Val/Val genotype, followed by the Val/Met genotype, and finally by the Met/Met genotype (Figure 3). As genotype does not change across cells, we can assume that this tendency is similar in the brain. Thus, it seems that incarceration does not induce epigenetic changes since the enzyme activity predicted by the genotype is effectively expressed by the individuals. These data are also in accordance with the literature (Tammimäki & Männistö, 2012; Weinshilboum & Dunnette, 2008).\n\nThe distribution of the COMT Val158Met polymorphism in our sample revealed values higher than those expected for Caucasian individuals regarding the Val/Val genotype, and lower than those expected for the Met/Met genotype (González-Castro et al., 2013). Nevertheless, due to the pattern of conduct, we can assume that our sample population does not represent a general population sample. In previous literature reports, the Val/Val genotype has been associated with the development of ASPD, in adults (Langley et al., 2010). Since ASPD is the only psychiatric disorder associated with premeditated aggression (Frick & White, 2008; Patrick, 2014), the higher prevalence of Val/Val genotype and the clear association of this genotype with premeditated aggression are logical. A possible explanation for these results could be the lower availability of dopamine, at the PFC level caused by Val/Val genotype (Matsumoto et al., 2003; Tunbridge, 2004). Lower availability of dopamine at the prefrontal level seems to be associated with higher ability to change mental representations and higher mental flexibility – yet, lower emotional sensitivity. This biochemical scenario can lead to higher ability to make action plans (Yildirim & Derksen, 2015).\n\nIn our study, genotype results were in good agreement with enzyme kinetic assays, in which we have found that higher erythrocyte S-COMT activity was also associated with premeditated aggression. In fact, while the value of KM was similar in both groups (since it is specific for the enzyme), individuals with premeditated aggression presented a Vmax value for S-COMT in erythrocytes twofold higher than those of the impulsive group, confirming that individuals with premeditated aggression have more available functional enzyme to convert the substrate (Figure 1).\n\nIn individuals with the Met/Met or Val/Met genotype, higher availability of dopamine at the PFC level (Matsumoto et al., 2003; Tunbridge, 2004) was already reported and seems to be associated with higher cognitive stability, less flexibility of mental representations, and higher emotional sensitivity (Yildirim & Derksen, 2015). This can be on the basis of the occurrence of impulsive aggression acts in individuals with lower erythrocyte S-COMT activity.\n\nAlthough it was neither an objective nor an initial hypothesis, the analysis of our data confirmed previous literature reports in which it is concluded that PCL-R can distinguish between premeditated and impulsive aggression (Blais et al., 2014). In addition, the activity of erythrocyte S-COMT was positively correlated with the PCL-R score (Figure 2). In other words, psychopathic traits measured by PCL-R were positively associated with erythrocyte S-COMT activity. This suggests that COMT activity can interfere with the phenotype of individuals with ASPD and psychopathy (Yildirim & Derksen, 2015). Thus, those who display higher COMT activity may be more emotionally insensitive and more prone to premeditated acts of aggression. Even though the correlation is weak, this biological finding has never been described and can be a starting point for future studies on a novel biological therapeutic target for a population that, so far, did not have relevant pharmacological interventions, besides symptomatic control (Bourne, 2010). Is COMT related to the antisocial personality? Can we interfere in personality through its pharmacological modulation? Although, psychiatrists are still cautious about the possibility of pharmacological or psychotherapeutic intervention in ASPD, this option may constitute a sound scientific basis with possible translation to clinical practice. Other authors have suggested that it is possible to change guilt-driven behaviour through the inhibition of COMT in healthy individuals (Mitchell, Weinstein, Vega, & Kayser, 2018; Sáez, Zhu, Set, Kayser, & Hsu, 2015).\n\nIn our sample, the DRD2/ANKK1 TaqIA polymorphism was not associated with different types of aggression. The prevalence of the allele A1 was lower than that described in Caucasian populations (Ma, Wang, Yuan, Su, & Li, 2015). According to previous findings, we expected higher prevalence of the DRD2 allele A1 in our sample, as the sample consists of offenders with significant substance use disorders (SUD) and psychopathic traits (Ponce et al., 2008; Wu & Barnes, 2013). Nevertheless, in our sample, the presence of DRD2 polymorphism did not seem to be differently associated to aggression types.\n\nThe therapeutic approach of aggression has been sustained by antipsychotics and mood stabilisers. However, a recent systematic review (Howner et al., 2020) highlights the lack of knowledge on the effectiveness of pharmacological treatment, within forensic psychiatry. These authors claimed that the frequent use of antipsychotics, sometimes in combination with other pharmacological agents, in this complex and heterogeneous patient group, calls for high-quality studies, in this specific setting. The present study suggests a biological difference in COMT activity between offenders with premeditated aggression and those with impulsive aggression. In fact, blockage of D2 and 5HT2 receptors can be useful for impulsive aggression, yet that is not the case for premeditated acts. Herein, we hypothesise that this type of aggression could be modulated with COMT inhibitors.\n\nConsidering the overall characterisation of our sample, we observed that it was mostly composed of individuals who had committed aggressive acts classified as impulsive. This type of aggression distribution is in accordance with that described for forensic populations (Swogger et al., 2015).\n\nThe forensic population described here presented an average age, education, and prevalence of violent crimes comparable with other forensic samples (Forrester, Till, Simpson, & Shaw, 2018). From a clinical point of view, we have verified that there was a high prevalence of ASPD and SUD. Other authors had previously reported high prevalence of ASPD and SUD in forensic populations (Widinghoff et al., 2019). The sample was collected from a forensic facility for convicted individuals who had committed multiple and recurrent crimes. This type of population, showing criminal recidivism, has a higher presence of psychopathic traits. In fact, PCL-R high scores, as the ones found in our sample, predict criminal recidivism (Olver & Wong, 2015).\n\nThe major limitations of our study are the small sample size, the fact that we have done multiple comparisons between the two groups, and those related to cross sectional studies. This design allowed to compare individuals with premeditated aggression with those with impulsive aggression, at a specific point in time, but we cannot exclude the possibility of low representativeness of the behaviour of the group as a whole (due to the fact that measurements are performed at a specific time point\|). Considering these limitations, our results are preliminary and shall be confirmed in larger populations. Even though, sample size shall be evaluated considering the particularities of this specific population and the limitations regarding the contact with patients, consultations, and research approval.\n\nIn conclusion, in male offenders, impulsive aggression may be associated with lower erythrocyte activity of S-COMT (confirmed by the Val/Val genotype of COMT). This confirms the correlation of COMT with aggression type, but not with its genesis. We believe that our preliminary results constitute a strong basis for future research with implications on the management of impulsive aggression in penitentiary institutions.\n\n\nData availability\n\nThe original contributions presented in the study are publicly available. This data can be found here: DOI: https://datadryad.org/stash/share/YbGXun9hQD_41NXFTx1MSlciIN3GEVqj4yt9WEu_598.\n\n\nAuthor contributions\n\nAll authors contributed to the study conception and design.\n\nConceptualisation: Jacinto Azevedo, Maria Vieira-Coelho, Rui Coelho, Margarida Figueiredo-Braga.\n\nData curation: Jacinto Azevedo.\n\nFormal analysis: Jacinto Azevedo.\n\nInvestigation: Jacinto Azevedo, Maria Augusta Vieira-Coelho, Cláudia Carvalho, Maria Paula Serrão, Margarida Figueiredo-Braga.\n\nMethodology: Jacinto Azevedo, Maria Vieira-Coelho, Rui Coelho, Margarida Figueiredo-Braga.\n\nProject administration: Jacinto Azevedo, Margarida Figueiredo-Braga, Maria Augusta Vieira-Coelho.\n\nResources: Jacinto Azevedo, Margarida Figueiredo-Braga, Maria Augusta Vieira-Coelho.\n\nSupervision: Maria Augusta Vieira-Coelho, Rui Coelho, Margarida Figueiredo-Braga.\n\nValidation: Maria Vieira-Coelho, Rui Coelho, Margarida Figueiredo- Braga.\n\nWriting – original draft: Jacinto Azevedo.\n\nWriting – review & editing: Jacinto Azevedo, Maria Vieira-Coelho, Rui Coelho, Margarida Figueiredo-Braga.\n\n\nCompeting interests\n\nThe authors have no conflict of interest to declare.", "appendix": "Acknowledgments\n\nWe would like to thank Letícia Malta, MD, and Nurse João Pinto for their collaboration in the acquisition of the data. 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Psychiatry. 2008; 193(2): 121–125. Publisher Full Text\n\nReidy DE, Krusemark E, Kosson DS, et al.: The Development of Severe and Chronic Violence Among Youth: The Role of Psychopathic Traits and Reward Processing. Child Psychiatry Hum. Dev. 2017; 48(6): 967–982. PubMed Abstract \| Publisher Full Text\n\nRivera-Iñiguez I, Panduro A, Ramos-Lopez O, et al.: DRD2/ANKK1 TaqI A1 polymorphism associates with overconsumption of unhealthy foods and biochemical abnormalities in a Mexican population. Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity. 2019; 24(5): 835–844. Publisher Full Text\n\nRosell DR, Siever LJ: The neurobiology of aggression and violence. CNS Spectr. 2015; 20(3): 254–279. Publisher Full Text\n\nSáez I, Zhu L, Set E, et al.: Dopamine modulates egalitarian behavior in humans. Curr. Biol. 2015; 25(7): 912–919. Publisher Full Text\n\nSiever LJ: Neurobiology of Aggression and Violence. Am. J. Psychiatr. 2008; 165(4): 429–442. Publisher Full Text\n\nStanford MS, Houston RJ, Mathias CW, et al.: Characterizing aggressive behavior. Assessment. 2003; 10(2): 183–190. Publisher Full Text\n\nSwogger MT, Walsh Z, Christie M, et al.: Impulsive versus premeditated aggression in the prediction of violent criminal recidivism. Aggress. Behav. 2015; 41(4): 346–352. Publisher Full Text\n\nTammimäki A, Männistö PT: Catechol-O-methyltransferase gene polymorphism and chronic human pain. Pharmacogenet. Genomics. 2012; 22(9): 673–691. Publisher Full Text\n\nTunbridge EM: Catechol-O-Methyltransferase Inhibition Improves Set-Shifting Performance and Elevates Stimulated Dopamine Release in the Rat Prefrontal Cortex. J. Neurosci. 2004; 24(23): 5331–5335. PubMed Abstract \| Publisher Full Text\n\nVieira-Coelho MA, Soares-da-Silva P: Effects of tolcapone upon soluble and membrane-bound brain and liver catechol-O-methyltransferase. Brain Res. 1999; 821(1): 69–78. Publisher Full Text\n\nVittinghoff E, McCulloch CE, Glidden DV, et al.: Linear and non-linear Regression Methods in Epidemiology and Biostatistics. Rao D, Miller CR, Rao JP, editors. Handbook of Statistics. Elsevier; 2007; (pp. 148–186). Publisher Full Text\n\nVolavka J, Laska E, Baker S, et al.: History of violent behaviour and schizophrenia in different cultures. Br. J. Psychiatry. 1997; 171(1): 9–14. PubMed Abstract \| Publisher Full Text\n\nvon Diemen L , Szobot CM, Kessler F, et al.: Adaptation and construct validation of the Barratt Impulsiveness Scale (BIS 11) to Brazilian Portuguese for use in adolescents. Revista Brasileira de Psiquiatria (Sao Paulo, Brazil: 1999). 2007; 29(2): 153–156. PubMed Abstract \| Publisher Full Text\n\nWeinshilboum R, Dunnette J: Thermal stability and the biochemical genetics of erythrocyte catechol-O-methyltransferase and plasma dopamine-β-hydroxylase. Clin. Genet. 2008; 19(5): 426–437. PubMed Abstract \| Publisher Full Text\n\nWidinghoff C, Berge J, Wallinius M, et al.: Gambling Disorder in Male Violent Offenders in the Prison System: Psychiatric and Substance-Related Comorbidity. J. Gambl. Stud. 2019; 35(2): 485–500. PubMed Abstract \| Publisher Full Text\n\nWitt K, van Dorn R , Fazel S: Risk Factors for Violence in Psychosis: Systematic Review and Meta-Regression Analysis of 110 Studies. PLoS One. 2013; 8(2): e55942. Publisher Full Text\n\nWolff N, Blitz CL, Shi J, et al.: Sexual Violence Inside Prisons: Rates of Victimization. J. Urban Health. 2006; 83(5): 835–848. PubMed Abstract \| Publisher Full Text\n\nWrangham RW: Two types of aggression in human evolution. Proc. Natl. Acad. Sci. 2018; 115(2): 245–253. PubMed Abstract \| Publisher Full Text\n\nWu T, Barnes JC: Two dopamine receptor genes (DRD2 and DRD4) predict psychopathic personality traits in a sample of American adults. J. Crim. Just. 2013; 41(3): 188–195. Publisher Full Text\n\nYildirim BO, Derksen JJL: Mesocorticolimbic dopamine functioning in primary psychopathy: A source of within-group heterogeneity. Psychiatry Res. 2015; 229(3): 633–677. PubMed Abstract \| Publisher Full Text" }	[ { "id": "285655", "date": "25 Jun 2024", "name": "Elias Abdalla-Filho", "expertise": [ "Reviewer Expertise Forensic Psychiatry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe text is written in a very clear and understandable way for the reader. Despite offering additional information to the objective of this study (suggesting new research), the authors were very objective and clear regarding the focus of this study: the assessment of dopaminergic function in incarcerated criminal individuals, studying the specification between COMT activity and the type of physical aggression, whether impulsive or premeditated. In different ways and even a little repetitively, it was clearly shown that erythrocyte S-COMT activity was significantly greater in aggressors with premeditated aggression than with impulsive aggression.\nThey also clearly described the limitations of this study, which, in turn, is only intended to be a preliminary study of this matter. As for bibliographical references, there is a large number of citations well before 5 years ago. However, the authors also brought information from current studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11894", "date": "28 Jun 2024", "name": "Jacinto Azevedo", "role": "Author Response", "response": "Dear Professor. Elias Abdalla-Filho, Thank you for your thorough and thoughtful review of our manuscript. We are pleased to receive your positive feedback and appreciate the time and effort you have taken to evaluate our work. We acknowledge your comments regarding the clarity and focus of our study, particularly the assessment of dopaminergic function in incarcerated criminal individuals and the differentiation between COMT activity in impulsive versus premeditated aggression. Your recognition of the significance of our findings and the clear presentation of our limitations is highly valued. Regarding your point on the bibliographical references, we appreciate your observation. We strived to balance seminal works with recent studies to provide a comprehensive background and context for our research. Moving forward, we will continue to incorporate the most current literature to enhance the relevance and timeliness of our references. We are grateful for your affirmation that our study design is appropriate, technically sound, and that we have provided sufficient methodological details for replication. Ensuring reproducibility and transparency is a priority for us, and your positive assessment in this regard is encouraging. Thank you once again for your valuable feedback and for approving our manuscript. We are confident that your expertise in forensic psychiatry has contributed to the robustness of our study, and we appreciate your support. Sincerely, Jacinto Azevedo" } ] }, { "id": "302289", "date": "08 Aug 2024", "name": "Mujgan Cengiz", "expertise": [ "Reviewer Expertise Molecular biology psychiatric 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\nThe study is well designed, well written, and its statistics are done, but the number of case used for the experiment is very low. It has been claimed that COMT may be associated with different types of aggression in violent offenders, although the writers accepted that number of cases is not sufficient to support this claim, we also thought the same thing. It is thought that it would not be appropriate for the number of cases and statistics to give a strong result, and therefore it is not possible to accept the article 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-224
https://f1000research.com/articles/10-1136/v1	10 Nov 21	{ "type": "Research Article", "title": "EMI radiation of power transmission lines in Malaysia", "authors": [ "Azhan Fikry", "Siow Chun Lim", "Mohd Zainal Abidin Ab Kadir", "Azhan Fikry", "Mohd Zainal Abidin Ab Kadir" ], "abstract": "Background: There has been rising concern amongst the public regarding their home's proximity to high tension power transmission lines. The primary cause of fear is the impact of the electromagnetic interference (EMI) radiation on the nearby occupants' health. Despite the presence of national permissible limits of EMI radiation, there is still lack of information with regards to the EMI radiation of the types of power lines configuration in Malaysia. Methods: The electric and magnetic fields of several selected power transmission lines were simulated using the EMFACDC software program from the recommendation ITU-T K.90. Five types of power transmission lines available in Malaysia are considered. Results: It was found that the simulated electric and magnetic field levels at all the power lines' right of way (ROW) boundary complies with the prescribed exposure limit. However, the electromagnetic fields (EMF) level increases significantly as the separation distance is reduced from 30m. For a more conservative approach, the ROW can be set at 30m across all transmission voltage level and corridor area condition. Conclusion: It can be concluded that Malaysia's power transmission lines are within the prescribed exposure limits. To further minimize the electric and magnetic field level, it is recommended that the residential building should be built at least 30 meters away from the power transmission lines, especially for the 275kV double circuit, 275/132kV quadruple circuit, and 500kV double circuit lines.", "keywords": [ "EMI radiation", "power transmission lines", "EMF", "ELF", "public exposure limit", "Right-of-Way", "ICNIRP" ], "content": "Introduction\n\nThe public is getting increasingly concerned about the potential biological and health effects of power transmission lines considering the risk of exposure to electromagnetic interference (EMI) radiation. Biological effects are noticeable responses to a stimulus or an environmental change.1 At an extreme level, electromagnetic fields (EMF) will affect humans' health, such as micro shocks and induced currents in the body.2 Power transmission lines produce electric and magnetic fields at an extremely low frequency. These transmission lines were sometimes located in close proximity to residential areas, which could potentially increase the exposure level of electromagnetic radiation. Although national and international guidelines have established exposure limits to protect the public against high-level EMF that might be harmful, there are still rising concerns among the public on whether their residency is affected by such radiation, especially to those living near power transmission lines.3\n\nOverhead power transmission lines in Malaysia are typically rated at 132 kV, 275 kV, and 500 kV. Figure 1 shows a typical dimension for various power transmission lines towers in Malaysia. These dimensions agree well with the transmission line design manual in I. Beck.4\n\nSeveral isolated simulation studies on the propagation of EMF around the 132kV, 275kV, and 500kV power transmission lines in Malaysia has been presented in Sahbudin et al.,5 Said and Hussain,6 Jimbin and Ahmad,7 and Rahman et al.8 All of the studies referenced here are related to EMF propagation around the power transmission lines and how it affects the nearby public. The studies were conducted in several locations in Malaysia in which the EMF level was analyzed and computed at a specific point and distance from the power transmission line. However, analysis of the compliance of the EMF with recommended public exposure limits remains lacking as no specific study evaluated the EMF level from every type of power transmission line in Malaysia. Hence, in this study, simulations of the electromagnetic field's radiation from various high-voltage power transmission lines in Malaysia are conducted simultaneously to provide an overall comparison in terms of their corresponding EMF level as a function of distance away from the power lines. The simulated EMF levels are then benchmarked with public exposure limits recommended by international standards and the commenced right of way (ROW), after which the minimum safe distance of power transmission lines from residential areas can then be determined.\n\n\nMethods\n\nFigure 2 depicts the overall project flow for this work.20 The field calculation for a three-phase circuit power transmission line is based on the principle of superposition.9,12 Suppose the three conductors are labelled with an index k that ranges from one to three. For each conductor, there is a combination of complex current Ik and complex voltage Vk. The position of the conductors is denoted by xkykin which the origin can be chosen at any point. The objective is to calculate the field at a point of interest (x, y). Every conductor is above the earth at the height of hk and rk away from the point of interest as shown in Figure 3. For a single conductor-carrying current I along a straight line, the magnetic field magnitude, B is given by:\n\nwhere μo: permittivity of free space.\n\nThe resultant magnetic field due to a three-phase conductor system is the complex summation of contributing magnetic field vectors. The vertical and horizontal components of B can then be established geometrically as shown in the National Grid EMF guide.13 Each conductor is then resolved into an in-phase and out-of-phase component to determine the current relation. This is done by referring to the phasor diagram depicted in the National Grid EMF guide.13 By referring to the phasor diagram, all the in-phase and out-of-phase components can be extracted using the sin and cos of 30° (120° − 90°). For a single three-phase circuit, it does not matter which order these conductors were placed. Each conductor will have a phase 120° apart from the other two conductors. Being 120° apart makes the phases balanced in which the power transfer is constant at any point. This method has also been used in other isolated simulation studies presented in Refs. 6 and 7. For more than one circuit, a consistent convention should be applied when defining the phases to achieve optimum efficiency as shown in the National Grid EMF guide.13\n\nBy multiplying both geometrical and current term, four components of the field can be obtained, which is Bin,x, Bin,y, Bout,x, and Bout,y. The total magnetic field is then given by:\n\nThe electric field generated by a system of conductors is attributable to the potential Vk applied to each of them. The electric coupling between conductors should be considered as well in order to determine the linear charge density.12 The potential V at its boundary surface for a single conductor of effective radius R' at a height h above the ground is given by:\n\nwhere λ: equivalent density of charge; ε0 is the permittivity of free space.\n\nThe conductor's electrical image's contribution was also incorporated in equation 3 in the case of perfectly conducting ground. For a known value of V, the equivalent linear density of charge can be assessed along with its electrical image, which is then used to calculate the electric field vector at any point of interest (x, y). A single contribution to the electric field either from the conductor or its electrical image is given by:\n\nwhere r: distance from the conductor (or image) to the point of interest; r̂ is the respective unit vector. In the event of multiple conductors (three conductors for a three-phase circuit), the equivalent linear density of charge is determined by solving the linear system of equations given by:\n\nwhereP: coupling matrix; λ: equivalent densities vector; V: vector of potentials applied to the conductors.\n\nThe coupling matrix can be defined as:\n\nHere the conductor indexes are denoted by i j from 1 to N. Dij represents the distance between the conductor i and the image of the conductor j while dij represents the distance between the conductor i and the conductor j. With V as the voltage level of the overhead power transmission lines in Malaysia as depicted in Figure 1, the charge density can be obtained by inverting the matrix equation above where:\n\nAfter solving the system of equations, the corresponding charge densities are then used to calculate the resultant electric field at the point of interest, as in the case of a single conductor described above. The resulting electric field due to a system of conductors (and their electric images) is obtained by the complex summation of the contributing electric field vectors.\n\nIn this study, the EMF simulations were conducted using the publicly available EMFACDC v2.0, update to Appendix II program developed by the Telecommunication Standardization Sector of International Telecommunication Union (ITU-T) under the series of recommendations known as Series K (https://www.itu.int/rec/T-REC-K.90-201905-I!Amd1/en). Table 1 shows the geometrical parameters of this study.\n\nVerification of the EMFACDC program is performed by comparing the simulation value obtained from the program with the value obtained through manual calculation performed for a typical power line setup. To do this, the exact parameter of the selected power line is keyed into the program. The results were considered satisfactory if both of the values were approximately similar to each other. This study chooses the magnetic field level of a typical United Kingdom (UK) 400kV double circuit power transmission line at 10 meters from the centerline. As this verification purpose is to test for the accuracy of the simulation program, any type of power line is applicable.\n\nRated current is used for each conductor in the power lines to test for the maximum current value possible as applied in Jimbin and Ahmad.7 The current in the left and right circuits are assumed to be equal in order to ensure the EMF level is balanced at the left and right ROW boundary. For each type of power transmission line, the conductors are represented by their phase pattern and assigned their own phase code. Standard transposed phasing and un-transposed phasing will be considered in this study as shown in Figures 4 and 5. Transposition of phasing is performed to reduce crosstalk or interference which arises by current flowing in the conductors. Therefore, un-transposed phasing is expected to produce the highest magnetic flux density and vice versa, as suggested in Said et al.,6 under Group 5 conductor phase pattern. Although transposed phasing is used by most of the power transmission lines in the National Grid to significantly reduce the magnetic and electric field generated, un-transposed phasing is still considered in this study as it is not always feasible to transpose every line.\n\nThe parameters for each type of power lines which are based on Malaysia’s specific power transmission line were consolidated from R. K. Z. Sahbudin et al.,5 I. Said et al.,6 V. S. Jimbin and Ahmad,7 N. A. Rahman et al.,8 I. M. Rawi et al.,10 and N. H. N. Hassan et al.11 These dimensions also agree well with the Transmission Line Design Manual prepared by R. W. Beck, Inc., for the use of Tenaga Nasional Berhad (TNB) in June 2000.4 Five types of power transmission lines available in Malaysia are considered in this study. These are the power transmission lines typically used in Malaysia, known with voltage levels of 132 kV, 275 kV, and 500 kV:\n\n1. 132 kV double circuit power transmission line.\n\n2. 275 kV double circuit power transmission line.\n\n3. 132/132 kV quadruple circuit power transmission line.\n\n4. 275/132 kV quadruple circuit power transmission line.\n\n5. 500 kV double circuit power transmission line.\n\nThese power transmission lines were identified based on the Suruhanjaya Tenaga Wayleave for Electricity Supply Lines.15 They were chosen as they were categorized under high voltage and extra-high voltage distribution and ran under three-phase configurations. Tables 2 and 3 show samples of the input data used for the field simulation of 275/132kV quadruple circuit power line and 500 kV double circuit power lines in Malaysia.7,8,10 The remaining set of input data i.e. of 132 kV double circuit power line, 275 kV double circuit power line and 132/132 kV quadruple circuit power line is made available in Tables 3, 4, and 5.20\n\nThe simulation of electric and magnetic fields at various horizontal distance from the center of power lines was analyzed and benchmarked with public exposure limits from selected organizations defined in Table 4 (Table 820). The International Commission on Non-Ionizing Radiation Protection (ICNIRP) and European Union (EU) limits were selected as they were used in most of the countries globally while Slovenia and Italy were purposely selected due to their more stringent requirement. In this study, public exposure limits from three different categories are chosen for the correlation studies to satisfy all variants in every country. The exposure limits selected were used to prevent established effects at high electromagnetic field level. The simulation is also benchmarked with the commenced ROW from Suruhanjaya Tenaga for each type of power lines to estimate the electromagnetic field level at the minimum separation distance from the center of power lines.15 The ROW specified by the Suruhanjaya Tenaga 15 is applicable for the correlation process as it is the currently commenced minimum safe distance for power transmission lines in Malaysia. Correlation between the exposure limit and the commenced ROW is analyzed by observing the electromagnetic field level at the minimum separation distance. The field level at the minimum separation distance is considered to be safe if it is lower than the exposure limits.\n\n\nResults\n\nThe simulation was initially verified with a typical setup of a double circuit power transmission line in the United Kingdom19 as outlined in the methods section (Table 220). Upon verification, simulation of magnetic flux densities and the electric field strength from different types of power lines at the horizontal line were conducted. The magnetic and electric field of the power transmission lines relative to the selected exposure limits and commenced ROW were summarized and shown in Tables 5 and 6 (Table 10 and Table 1120).\n\nThe magnetic field due to all the power transmission lines are within the exposure limits set by the organizations in Table 4 (Table 820). The highest magnetic flux densities at the ROW boundary were simulated from the un-transposed 500kV double circuit power line under non-conducting ground condition as shown in Table 10.20 Even directly below the overhead power lines, the magnetic field levels are still within the exposure limit recommended by ICNIRP and EU.16,17\n\nSimilarly, the electric field due to all the power transmission lines are within with the exposure limits set by ICNIRP and EU. However, 25% of the power transmission lines exceed the stricter exposure limit practiced in Slovenia and Italy. The highest electric field strength data at the ROW boundary was simulated from the 500kV double circuit power line under un-transposed and conducting ground condition (Table 1120). Similar to the magnetic field simulation, even below the overhead power lines, the electric field levels are still within the exposure limit recommended by ICNIRP 2010 and EU Recommendation 1999.16,17 The transposed conductors give significantly lower magnetic flux densities and electric field strength.\n\n\nConclusions\n\nThe electric and magnetic field due to five types of power transmission lines in Malaysia were simulated and benchmarked with the known exposure limits and commenced ROW. The electric and magnetic field level for all types of power lines at the edge of ROW was found to be lower than the exposure limit recommended by ICNIRP. Hence, it can be concluded that Malaysia's power transmission lines are within the safe exposure limit recommended by ICNIRP. To further minimize the electric and magnetic field level, it is recommended that the residential building should be built at least 30 meters away from the power transmission lines, especially for the 275kV double circuit, 275/132kV quadruple circuit, and 500kV double circuit lines.\n\n\nSoftware availability\n\nThe software EMFACDC v2.0, appendix 1 (2020) which is used in simulating the electric and magnetic field in this study is publicly available to download at https://www.itu.int/rec/T-REC-K.90-201905-I!Amd1/en.\n\n\nAuthor roles\n\nAzhan F.: Conceptualization, Formal Analysis, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing;\n\nSiow C.L.: Conceptualization, Supervision, Writing – Review & Editing\n\nM. Zainal A.A.K.: Conceptualization, Writing – Review & Editing\n\n\nData availability statement\n\nFigshare: Data for Electric and Magnetic Field of Power Transmission Lines in Malaysia.\n\nhttps://doi.org/10.6084/m9.figshare.16577423.v2.20\n\nThe project contains the following underlying data:\n\n• Data for Electric and Magnetic Field of Power Transmission Lines in Malaysia. (Simulated dataset of electric and magnetic field of five types of power transmission lines in Malaysia in Tables 1-12 and Figures 1-20).\n\n• Figure 1. (Overhead power transmission lines towers in Malaysia).\n\n• Figure 2. (Overall project flow).\n\n• Figure 3. (Geometrical illustration for the magnetic and electric field calculation).\n\n• Figure 4. (Double circuit phase configuration for standard transposed phasing (left) and un-transposed phasing (right).\n\n• Figure 5. (Quadruple circuit phase configuration for standard transposed phasing (left) and un-transposed phasing (right).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to thank the International Trade Union (ITU) for providing the EMFACDC v2.0, appendix 1 (2020) software and the Faculty of Engineering, Multimedia University (MMU) for supporting this study.\n\n\nReferences\n\nWorld Health Organization (WHO): Radiation: Electromagnetic fields. Aug. 04, 2016. (Accessed Feb. 10, 2021).Reference Source\n\nDepartment of Energy & Climate Change:Power Lines: Control of Microshocks and other indirect effects of public exposure to electric fields A voluntary Code of Practice.no. July, 2013.\n\nFarag AS, Cheng TC, Penn D: Development of an electromagnetic fields risk communication plan. Electr. Power Syst. Res. 1999; 50(1): 55–63. Publisher Full Text\n\nU.S. Department of Commerce: I. Beck (RW), “Tenaga Nasional Berhad Transmission Line Design Manual. Final Submittal.\|National Technical Reports Library - NTIS. National Technical Reports Library. 2000. (accessed Feb. 11, 2021).Reference Source\n\nSahbudin RKZ, Fauzi SA, Hitam S, et al.: Investigation of electrical potential and electromagnetic field for overhead high voltage power lines in Malaysia. J. Appl. Sci. 2010; 10(22): 2862–2868. Publisher Full Text\n\nSaid I, Hussain HB: Computation of magnetic field from quadruple tower transmission lines in Malaysia. Proc. Univ. Power Eng. Conf. 2008. Publisher Full Text\n\nJimbin VS, Ahmad NA: Magnetic field measurement from 132/275 kV overhead power lines. J. Teknol. Jan. 2017; 79(1): 89–95. Publisher Full Text\n\nRahman NA, Mahadi WNL, Said I, et al.: Simulation Approach in Evaluating the Electromagnetic fields from the new lines of Extra High Voltage (EHV) circuits near residential area in Malaysia. Eur. J. Sci. Res. 2010; 40(2): 189–198.\n\nIsmail HM: Characteristics of the magnetic field under hybrid ac/dc high voltage transmission lines. Electr. Power Syst. Res. 2009; 79(1): 1–7. Publisher Full Text\n\nRawi IM, Abidin Ab Kadir MZ, Gomes C, et al.: A case study on 500 kV line performance related to lightning in Malaysia. IEEE Trans. Power Deliv. Oct. 2018; 33(5): 2180–2186. Publisher Full Text\n\nHassan NHN, Bakar AHA, Mokhlis H, et al.: Analysis of arrester energy for 132kV overhead transmission line due to back flashover and shielding failure. PECon 2012-2012 IEEE Int. Conf. Power Ener. 2012; 1(December): 683–688. Publisher Full Text\n\nInternational Telecommunication Union (ITU): ITU-T Evaluation techniques and working procedures for compliance with exposure limits of network operator personnel to power-frequency electromagnetic fields Recommendation ITU-T K.90. 2018.Reference Source\n\nNational Grid EMF: How to calculate the magnetic field from a three-phase circuit. (accessed Feb. 11, 2021).Reference Source\n\nIEEE: IEEE Std C37.1 - Standard Definition, Specification, and Analysis of Systems Used for Supervisory Control, Data Acquisition, and Automatic Control. 1994; vol. 1994.\n\nEnergy Commission of Malaysia: Wayleave for Electricity Supply Lines: Your Right and Safety. 2014.\n\nInternational Commission on Non-Ionizing Radiation Protection (ICNIRP): ICNIRP Publication 2010, “ICNIRP Guidelines for Limiting Exposure to Time-Varying Electric and Magnetic Fields (1 Hz-100 kHz) International Commission on Non-Ionizing Radiation Protection*,”.2010. Publisher Full Text\n\nThe Council Of The European Union (CEU): Council Recommendation of 12 July 1999 on the limitation of exposure of the general public to electromagnetic fields (0 Hz to 300 GHz). Off. J. Eur. Communities. 1999; 59–70.\n\nWorld Health Organization (WHO): GHO\|By category\|Exposure limits for low-frequency fields (public) - Data by country. WHO.\n\nNational Grid EMF: Geometries of power lines.(accessed Feb. 11, 2021).Reference Source\n\nChun Lim S, Fikry A, Ab-Kadir MZA: Data for Electric and Magnetic Field of Power Transmission Lines in Malaysia. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "120587", "date": "07 Feb 2022", "name": "Jacek Paś", "expertise": [ "Reviewer Expertise Electronic safety systems", "compatibility" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease add at the end of the article a list of abbreviations used in the article.\n\nThe critical review of the literature on the state of the issue is done poorly.\n\nPlease expand the conclusions in the article, they are currently only four sentences long. Not all important graphs are included in the conclusions.\n\nThe article and low frequency electromagnetic field calculations around power lines are very interesting, and the results can be used to determine safety zones.\n\nThe issue of low-frequency electromagnetic field effects is important to the health of people living near power lines. Please refer to the E, H component limits of standards in other countries, e.g. USA, European countries and the proposed regulations 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7846", "date": "24 Feb 2022", "name": "Chun Lim Siow", "role": "Author Response", "response": "Thank you for your invaluable comments to improve this article. The improvements made are summarised below: Please add at the end of the article a list of abbreviations used in the article. The list of abbreviations has been added at the end of the article. The critical review of the literature on the state of the issue is done poorly. The literature review has been improved. The contributions from related and cited articles have been discussed and the research gap has been clearly identified and stated. Please expand the conclusions in the article, they are currently only four sentences long. Not all important graphs are included in the conclusions. The conclusion has been expanded to be more comprehensive. The article and low frequency electromagnetic field calculations around power lines are very interesting, and the results can be used to determine safety zones. Yes, this is indeed an important issue where public awareness and understanding still has a lot of room for improvement. The issue of low-frequency electromagnetic field effects is important to the health of people living near power lines. Please refer to the E, H component limits of standards in other countries, e.g. USA, European countries and the proposed regulations in the country. Thank you for the recommendation. The low frequency electromagnetic field effect is indeed a very important issue for the health of people living near power lines. In this paper, we have benchmarked against the world’s largest and most relevant organization namely ICNIRP, which is collaborating with many other relevant stakeholders such as World Health Organisation and national radiation protection bodies at the global level. We have also benchmarked against a regional recommendation which is European Union and two European countries (Slovenia and Italy) which sets more stringent exposure limits for the E and H components. For future work, it would be interesting to consider USA (with so many large states) and other European countries as per your recommendation." } ] }, { "id": "99796", "date": "07 Feb 2022", "name": "Muhammad Ammirrul Atiqi Mohd Zainuri", "expertise": [ "Reviewer Expertise Power Electronics", "Power Quality", "Electrical Power Engineering", "Renewable Energy System", "Artificial Intelligence and Agriculture-Photovoltaic." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction:\nAdd proper objectives like \"this paper or manuscript present... \"\n\nAdd also an overview of each section at the end of the introduction part.\n\nMethods:\n\nExplain in more detail the flowchart of Figure 2.\nResults:\nDo you have any waveform that can show the EMI related to Electric field strength?\n\nMore discussion needed as referred to Table 6 in reference to each parameter discussed as in table 6.\n\nConclusions:\nToo short, add more summary on the methods and results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7847", "date": "24 Feb 2022", "name": "Chun Lim Siow", "role": "Author Response", "response": "Thank you for the invaluable comments to improve this paper. The improvements made are summarised below: Introduction: Add proper objectives like \"this paper or manuscript present... \" The objective has been rephrased as follows: Hence, this paper presents the simulations of the electromagnetic field radiation from various high-voltage power transmission lines in Malaysia conducted simultaneously to provide an overall comparison in terms of their corresponding EMF level as a function of distance away from the power lines. Add also an overview of each section at the end of the introduction part. The overview of each section has been added at the end of the introduction part. Methods: Explain in more detail the flowchart of Figure 2. Flowchart of Figure 2 has been explained in more detail under the Methods section. Results: Do you have any waveform that can show the EMI related to Electric field strength? An additional Reference 21 has been cited to support the relation between EMI and electric field strength. More discussion needed as referred to Table 6 in reference to each parameter discussed as in table 6. The discussion on Table 6 has been extended Conclusions: Too short, add more summary on the methods and results. The conclusion has been expanded to be more comprehensive." } ] } ]	1	https://f1000research.com/articles/10-1136
https://f1000research.com/articles/11-221/v1	23 Feb 22	{ "type": "Brief Report", "title": "Effect of hydrothermal treatment of titanium in high concentration of AgNO3 solution on surface morphology and roughness", "authors": [ "Sunarso Sunarso", "Raihan Jazmi Hares Putra", "Citra Fragrantia Theodorea", "Azizah Intan Pangesty", "Raihan Jazmi Hares Putra", "Citra Fragrantia Theodorea", "Azizah Intan Pangesty" ], "abstract": "Development of silver (Ag) modified titanium (Ti) as an antibacterial dental implant has recently been growing. Ag demonstrated an excellent antibacterial property without the risk of bacterial resistance. Hydrothermal treatment using AgNO3 solution is one of the facile and promising methods to modify Ti surface with Ag. However, the effect of high AgNO3 concentration and the absent of a toxic reduction agent has not been clearly studied. In this study, Ti surface was hydrothermally treated in 0.01 mol/L and 0.1 mol/L AgNO3 solutions at 150oC for 24 hours. Analysis of surface morphology using scanning electron microscopy with energy dispersive X-ray analysis suggested the formation of non-homogenous Ag coating with a tendency to be aggregated and thicken with the increase of AgNO3 concentration. The Ag coating deposited on Ti surface were composed of mainly metallic and some oxide forms. Surface roughness of all AgNO3 treated Ti surface was comparable based on the analysis of surface roughness parameter. In conclusion, hydrothermal treatment of Ti surface in solely AgNO3 solution at high concentration produced non-homogenous Ag coating on its surface without significantly changed surface roughness. Keywords: Silver nitrate, titanium, hydrothermal, surface morphology, roughness", "keywords": [ "Silver nitrate", "titanium", "hydrothermal", "surface morphology", "roughness" ], "content": "Introduction\n\nTitanium (Ti) has widely been used clinically for dental implants due to their excellent mechanical properties, biocompatibility and osteoconductivity.1,2 Development of Ti implant for dental application is still challenging. Dental implant not only requires osseointegration capability but also antibacterial property.3 Titanium demonstrated satisfactory osseointegration clinically. However, its antibacterial capability is lacking.\n\nSilver coating has emerged as an alternative to prepare antibacterial titanium surface.4 Silver is considered a promising element to prevent and combat implant-related infection. Its main advantage is that it would not induce bacterial resistance.5 Silver is coated onto Ti surface often in the form of particles by immersion in AgNO3 solution mixed with a reduction agent.6,7 This results in silver particulates being deposited on the Ti surface often in nanoscale, thus called silver nanoparticles (AgNPs). The use of reduction agents is problematic because they are often toxic chemicals such as Sodium borohydride, ammonium formate and hydrazine.8,9 A recent study has been conducted to coat Ti surface with silver under hydrothermal without the need for reduction agents.10\n\nDirect Ag coating onto Ti surface using hydrothermal has not been clearly described especially in high concentration of AgNO3 solutions and without the addition of toxic reduction chemicals. Therefore, this study aimed to coat the Ti surface with Ag particles using hydrothermal using solely AgNO3 solution. Surface morphology including the distribution of the Ag coating and the change in surface roughness were then evaluated.\n\n\nMethods\n\nTwo Ti plates (Maximus Guard, Tokopedia) with a size of 10 cm × 10 cm and thickness of 1 mm were cut into 10 mm × 10 mm using a diamond cutter. A total of fifteen Ti plates (10 mm × 10 mm) were used in this study. The samples were washed ultrasonically with acetone, ethanol, and distilled water before drying. Silver nitrate (AgNO3, Merck) solutions with a concentration of 0.01 mol/L and 0.1 mol/L were prepared. Titanium samples were immersed in a 100 ml-size Teflon container with 25 ml of AgNO3 solutions, which was then placed into a hydrothermal vessel (FBA_Lab, Tokopedia). The hydrothermal vessel was heated in an oven at 150oC for 24 hours. After hydrothermal treatment, the samples were washed with ethanol three times before drying.\n\nThe elemental composition of titanium surface samples was examined using energy dispersive spectroscopy (Oxford instruments, UK) and analyzed using Oxford Aztect software. Scanning electron microscope (SEM) (Thermoscientific Quanta 650) (accelerating voltage (HV): 12kV, Secondary electron (SE), working distance (WD): 10.3-10.4mm) was used to evaluate the morphology of surface before and after hydrothermal. Surface roughness of titanium samples before and after hydrothermal treatment are measured using roughness tester (Surtronic S128) (Sampling length (l): 7 mm, cut-off (λc)/Type: 0.25 mm/2CR, Range: 100 μm). The average values of surface roughness were calculated using Microsoft excel spreadsheet software.\n\n\nResults and discussion\n\nFigures 1 and 2 show the photographs and SEM images of Ti surface before and after hydrothermal in AgNO3 solutions. Bright particles were observed on Ti surface hydrothermally treated in 0.01 mol/L and 0.1 mol/L AgNO3. An area showed more concentrated particles, which may indicate the agglomeration. At higher concentration of AgNO3 (0.1 mol/L), that concentrated area was larger (Figure 2). The elemental analysis from the surface using energy dispersive X-ray analysis (EDX) indicated that those particles are Ag (Figures 3-5). Many methods have been developed to coat Ag into Ti surface both in the form of particles or ions.4,11\n\nThe formation of silver particles on Ti surface under hydrothermal from AgNO3 solution was still unclear in this study. Several different mechanisms may be responsible for how Ag could be deposited on Ti surface from AgNO3 under hydrothermal treatment. One possible way is through thermal decomposition.12 The deposition of Ag particles from only AgNO3 solution up to 75 μmol/L under hydrothermal conditions was recently reported.10 However, the exact mechanism on how Ag particles could be deposited on Ti surface was not clearly described. AgNO3 was also reported to transform into Ag nanoparticles under hydrothermal condition at 121oC.13 Hydroxyl ions exist on Ti oxide layer may also play a role on the Ag particles growth on its surface.6 It is known that Ti surface naturally forms thin oxide layer which contain Ti-OH groups on its outmost part. Figure 2 has confirmed the formation of Ag particles on Ti surface both in 0.01 mol/L and 0.1 mol/L AgNO3 solutions. The Ag particles were also observed in the solution after hydrothermal (solution turned darkish color). The Ag particles seem to be non-homogenously distributed on Ti surface. As explained above, there is an area which contain thicker aggregated Ag particles masking the Ti surface. The concentrated Ag area was found to be larger in 0.1 mol/L AgNO3 than that in 0.01 mol/L AgNO3. These findings suggest that at high concentration of AgNO3, the Ag coating tends to be aggregated and thicker, thus the use of a lower concentration might be preferred.\n\nThe next question is that whether the Ag coating deposited on Ti surface is in the metallic or oxide forms. One way to find this is using the EDX elemental mapping to the aggregated Ag coating. Figures 3 and 4 show the elemental mapping of Ag coated Ti prepared from 0.01 mol/L and 0.1 mol/L AgNO3 solutions.14 In Figure 3, a thick Ag coating area (white dash line) shows a very strong purple color compared to the area in which less Ag coating was observed (white arrows). In Ti and oxygen (O) element mapping (green color and yellow color respectively), the Ag coating area was darker compared to the rest. The O elemental mapping provided very important data about the deposited Ag coating. The darker area (white dash line; Figure 3) in O elemental mapping indicated that that area was composed mostly of metallic Ag. Ag oxide also existed since the bright yellow color was also observed sporadically inside the white dash line (Figure 3; O Kα1). A similar trend was shown in Figure 4 where the Ag aggregate coating is larger. The thick Ag coating was most likely composed mainly from metallic Ag and smaller portion of Ag2O\n\nSurface treatment often changes surface roughness.15 The change in surface roughness might alter the biological performance of Ti implant. Therefore, it is necessary to evaluate whether the current method of Ag coating changed the surface roughness of Ti surface. Surface roughness parameters roughness average (Ra), maximum profile peak height (Rp), maximum profile valley depth (Rv), and mean roughness depth (Rz) were measured from all sample surfaces. Surface roughness texture of the sample surfaces were shown15 in Figure 6.16 The surface texture of all Ti samples before and after Ag coating were comparable. This data suggests that no significant changes were observed on Ti surface after Ag coating (Table 1). Comparison of SEM images between Ag coating and untreated Ti surfaces (Figure 2) also support surface texture data. The Ti substrate in which Ag coating deposited was found to be comparable (Figure 2A and B).\n\nThe values were calculated from three independent samples.16\n\nTable 1 shows the quantitative values of surface parameter obtained from the roughness tester. The surface Ra and Rv values of all samples were relatively similar which indicating its comparable average surface height and deepest valley of the substrate. A slight increase of Rp and Rz values were recorded in Table 1. Surface roughness Rp shows the maximum peak height which come from the Ag coating aggregate and was larger when a solution of 0.1 mol/L AgNO3 was used. The slight increase of Rp was followed by slight increase of Rz. Taken together, all surface roughness parameter demonstrated comparable values between untreated Ti (UnTi) and Ag coated Ti samples. This result suggests that hydrothermal treatment of Ti in 0.1 mol/L (Ag-Ti 0.1) and 0.01 mol/L AgNO3 (Ag-Ti 0.01) did not cause a notable change in the surface roughness.\n\n\nConclusion\n\nThis study reported the effect of hydrothermal treatment of Ti surface in AgNO3 solution on the surface morphology and roughness. After hydrothermal treatment, an Ag coating was observed in all treated Ti surfaces. EDX mapping suggests that Ag coating composed of mainly metallic Ag and in smaller quantities, Ag oxide. Using a higher AgNO3 solution concentration resulted in more Ag aggregates that mask Ti surface, creating a non-homogenous coating. Surface roughness of treated Ti surface did not change significantly when coated. Nevertheless, a slight increase of Rp and Rz was observed; this might be due to Ag coating aggregates.\n\n\nData availability\n\nFigshare: SEM and EDX https://doi.org/10.6084/m9.figshare.1715923414\n\nThis project contains the following underlying data:\n\n- RAW SEM image (Fig 2A).jpg\n\n- RAW SEM image (Fig 2B).jpg\n\n- RAW SEM image (Fig 2C).jpg\n\n- RAW SEM image (Fig 2D).jpg\n\n- RAW SEM image (Fig 2E).jpg\n\n- RAW SEM image (Fig 2F).jpg\n\n- RAW EDX Mapping Data for 0.01M AgNO3 treated Ti (Fig. 3)\n\n- RAW EDX Mapping Data for 0.1M AgNO3 treated Ti (Fig. 4)\n\n- RAW EDX Map sum element spectrum for untreated Ti (Fig. 5A)\n\n- RAW EDX Map sum element spectrum for 0.01M treated Ti (Fig. 5B)\n\n- RAW EDX Map sum element spectrum for 0.1M treated Ti (Fig. 5C)\n\nFigshare: Surface roughness https://doi.org/10.6084/m9.figshare.1721595816\n\nThis project contains the following underlying data:\n\n- Output files for roughness testing\n\no 0.01 M-1_1.jpg, 0.01 M-1_2.jpg, 0.01 M-1_3.jpg, 0.01 M-1_4.jpg (Roughness testing for 0.01 M AgNO3 treated Ti (specimen 1))\n\no 0.01 M-2_1.jpg, 0.01 M-2_2.jpg, 0.01 M-2_3.jpg, 0.01 M-2_4.jpg (Roughness testing for 0.01 M AgNO3 treated Ti (specimen 2))\n\no 0.01 M-3_1.jpg, 0.01 M-3_2.jpg, 0.01 M-3_3.jpg, 0.01 M-3_4.jpg (Roughness testing for 0.01 M AgNO3 treated Ti (specimen 3))\n\no 0.1 M-1_1.jpg, 0.1 M-1_2.jpg, 0.1 M-1_3.jpg, 0.1 M-1_4.jpg (Roughness testing for 0.1 M AgNO3 treated Ti (specimen 1))\n\no 0.1 M-2_1.jpg, 0.1 M-2_2.jpg, 0.1 M-2_3.jpg, 0.1 M-2_4.jpg (Roughness testing for 0.1 M AgNO3 treated Ti (specimen 2))\n\no 0.1 M-3_1.jpg, 0.1 M-3_2.jpg, 0.0 M-3_3.jpg, 0.1 M-3_4.jpg (Roughness testing for 0.1 M AgNO3 treated Ti (specimen 3))\n\no UnTi 1_1.jpg, UnTi 1_2.jpg, UnTi 1_3.jpg, UnTi 1_4.jpg (Roughness testing for untreated Ti (specimen 1))\n\no UnTi 2_1.jpg, UnTi 2_2.jpg, UnTi 2-2_3.jpg, UnTi 2_4.jpg (Roughness testing for untreated Ti (specimen 2))\n\no UnTi 3_1.jpg, UnTi 3_2.jpg, 0.0 UnTi 3_3.jpg, UnTi 3_4.jpg (Roughness testing for untreated Ti (specimen 3))\n\nFigshare: SEM and EDX https://doi.org/10.6084/m9.figshare.1715923414\n\nThis project contains the following extended data:\n\n- Photograph Fig 1.jpg\n\nRAW EDX Mapping for untreated Ti\n\nFigshare: Surface roughness https://doi.org/10.6084/m9.figshare.1721595816\n\nThis project contains the following extended data:\n\n- Summary roughness.xlsx (Aggregated data from surface roughness testing)\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\nConceptualization and methodology, S.; validation, S.; investigation, R.J.H.P.; resources, S.; writing—original draft preparation, S.; writing—review and editing, S., C.F.T. and A.I.P.; visualization, S.; supervision, S. and A.I.P.; funding acquisition, S. and A.I.P.", "appendix": "References\n\nAnanth H, Kundapur V, Mohammed HS, et al.: A review on biomaterials in dental implantology. Int. J. Biomed. Sci. 2015; 11: 113–120.\n\nBrånemark PI: Osseointegration and its experimental background. J. Prosthet. Dent. 1983; 50: 399–410. Publisher Full Text\n\nChen Z, Wang Z, Qiu W, et al.: Overview of Antibacterial Strategies of Dental Implant Materials for the Prevention of Peri-Implantitis. Bioconjug. Chem. 2021; 32(4): 627–638. PubMed Abstract \| Publisher Full Text\n\nJuan L, Zhimin Z, Anchun M, et al.: Deposition of silver nanoparticles on titanium surface for antibacterial effect. Int. J. Nanomedicine. 2010; 5: 261–267. Publisher Full Text\n\nWang H, Wang M, Xu X, et al.: Multi-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance. Nat. Commun. 2021; 12(1): 3331. PubMed Abstract \| Publisher Full Text\n\nFerraris S, Spriano S, Miola M, et al.: Surface modification of titanium surfaces through a modified oxide layer and embedded silver nanoparticles: Effect of reducing/stabilizing agents on precipitation and properties of the nanoparticles. Surf. Coat. Technol. 2018; 344: 177–189. Publisher Full Text\n\nGaviria J, Alcudia A, Begines B, et al.: Synthesis and deposition of silver nanoparticles on porous titanium substrates for biomedical applications. Surf. Coat. Technol. 2021; 406: 126667. Publisher Full Text\n\nPrabhu S, Poulose EK: Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Int. Nano Lett. 2012; 2(1): 32. Publisher Full Text\n\nIjaz Hussain J, Kumar S, Adil Hashmi A, et al.: Silver nanoparticles: preparation, characterization, and kinetics, Advanced. Mater. Lett. 2011; 2(3): 188–194. Publisher Full Text\n\nMohandas A, Krishnan AG, Biswas R, et al.: Antibacterial and cytocompatible nanotextured Ti surface incorporating silver via single step hydrothermal processing. Mater. Sci. Eng. C. 2017; 75: 115–124. PubMed Abstract \| Publisher Full Text\n\nFunao H, Nagai S, Sasaki A, et al.: A novel hydroxyapatite film coated with ionic silver via inositol hexaphosphate chelation prevents implant-associated infection. Sci. Rep. 2016; 6(1): 23238. PubMed Abstract \| Publisher Full Text\n\nNavaladian S, Viswanathan B, Viswanath RP, et al.: Thermal decomposition as route for silver nanoparticles. Nanoscale Res. Lett. 2006; 2(1): 44–48. PubMed Abstract \| Publisher Full Text\n\nAhmadi O, Jafarizadeh-Malmiri H, Jodeiri N: Optimization of Processing Parameters for Hydrothermal Silver Nanoparticles Synthesis Using Aloe vera Leaf Extract and Estimation of their Physico-Chemical and Antifungal Properties. Z. Phys. Chem. 2019; 233(5): 651–667. Publisher Full Text\n\nSunarso S: SEM and EDX. figshare. Dataset. 2022. Publisher Full Text\n\nZareidoost A, Yousefpour M, Ghaseme B, et al.: The relationship of surface roughness and cell response of chemical surface modification of titanium. J. Mater. Sci. Mater. Med. 2012; 23(6): 1479–1488. Publisher Full Text\n\nSunarso S: Surface roughness. figshare. Dataset. 2022. Publisher Full Text" }	[ { "id": "124776", "date": "01 Apr 2022", "name": "Le Thi Bang", "expertise": [ "Reviewer Expertise Biomaterials", "calcium phosphate based bioceramic", "metal implant", "surface modification" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research work is the investigation of Ti surface morphology and topography under hydrothermal treatment of Ti in solely AgNO3 at different concentrations for the antibacterial application. Method in this study is facile and effective for surface modification of any implant shape. The results showed that Ag coating composed of mainly metallic Ag and in smaller quantities, Ag oxide with higher agglomeration at higher Ag concentrations. And the coating does not alter the surface roughness significantly.\nThe study design is appropriate and the work is technically sound. However, some aspects have to be addressed:\nThere are several grammatical errors needed to be revised, for some examples:\n“the absent of a toxic reduction…” should be “the absence of….”\n“This results in silver particulates being deposited on the Ti surface often in nanoscale, thus called silver nanoparticles (AgNPs)”- verb of the sentence is missing.\n“Figures 1 and 2 show the photographs and SEM images” should be “Figures 1 and 2 show the photographs and SEM images, respectively”. In addition, please give some explanation in the text for Fig. 1.\n“The concentrated Ag area was found to be larger in 0.1 mol/L AgNO3 than that in 0.01 mol/L AgNO3. These findings suggest…” should be “The concentrated Ag ,,,, this finding suggests…”\nFurther comments:\nAlthough the coating layer was analyzed by the EDX mapping, and it seems that the author speculates the phase of the coating. Do the authors confirm the phase identification of the Ti sample and the precipitated powder after hydrothermal treatment by XRD?\n\nWhy is the reduction agent needed to be used for the silver nanoparticle coating in previous studies? For example, for a homogeneous coating? What is the positive effect of using solely AgNO3 coating to produce the antibacterial property to the Ti implant in comparison to the previous study? In Page 2 the authors stated that “It is known that Ti surface naturally forms thin oxide layer which contain Ti-OH groups on its outmost part” please revise this because the negative charge of Ti-OH was only obtained after treatment in alkali solution (see 1)? Did the coating particles just precipitate on the Ti surface or there is any possibility of bonding between the coating and the substrate? In the SEM of the coating sample, the coating particles are not in nanoscale size? Can the author give some possibility of agglomeration of the particles? In page 3, the author stated that “The concentrated Ag area was found to be larger in 0.1 mol/L AgNO3 than that in 0.01 mol/L AgNO3. These findings suggest that at high concentration of AgNO3, the Ag coating tends to be aggregated and thicker, thus the use of a lower concentration might be preferred” please explain why? What is the minimum amount Ag requirement for the antibacterial effect and the maximum Ag amount that is safe for the application? Do the homogenous and entirely cover coating is needed for the application?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "154122", "date": "17 Nov 2022", "name": "John Nicholson", "expertise": [ "Reviewer Expertise Dental materials", "oral biocompatibility", "alloys", "cements." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper reports a useful and well-planned study of the effect of hydrothermal treatment of titanium with silver nitrate, with the aim of developing a new type of implant surface with inherent anti-bacterial properties.\nSurface analysis of the finished metals showed that a non-homogeneous silver coating is deposited on the titanium, which is thicker with higher concentrations of silver nitrate in the treatment solution. Roughness of the surface was almost unchanged by the treatment. The finished coating is a potentially useful modification for clinical application in dental implants.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-221
https://f1000research.com/articles/10-442/v1	03 Jun 21	{ "type": "Correspondence", "title": "Comments about the appraisal of systematic reviews in restorative dentistry", "authors": [ "Kelvin I. Afrashtehfar" ], "abstract": "Adequate adoption of evidence-based practice is deeply rooted in accessing methodological quality and completeness of systematic reviews and meta-analyses reporting. Nonetheless, this assumption might be flawed if the methodological quality assessment has not been properly conducted. Taking the former statement into consideration, this correspondence article encourages the improvement of future methodological quality assessment manuscripts, especially in the field of restorative dentistry. Thus, this article addresses a methodological quality assessment about systematic reviews in restorative dentistry by Sarkis-Onofre et al. in the May 2019 issue of the Journal of Esthetic and Restorative Dentistry as an example of evaluating appraisals of reviews for increasing the awareness of reviewers, authors, and readers.", "keywords": [ "data management", "dentistry", "evidence-based medicine", "evidence-based practice" ], "content": "Comments about the appraisal of systematic reviews in restorative dentistry\n\nDear respectable Advisory Editors and readers:\n\nI read with great interest the publication by Sarkis-Onofre et al.1 “Systematic reviews in restorative dentistry: discussing relevant aspects,” in the Journal of Esthetic and Restorative Dentistry in the May 2019 issue. This well-written methodological quality assessment of systematic reviews or systematic review of systematic reviews stated that “This study was not registered in PROSPERO” since PROSPERO indicates that “Reviews of methodological issues need to contain at least one outcome of direct patient or clinical relevance in order to be included in PROSPERO.” Interestingly, despite the fact that the above referenced review falls in the PROSPERO's review of reviews category, it was neglected to being classified as such. Therefore, the authors' arguments not to register their protocol in PROSPERO are not valid.\n\nMoreover, the authors1 mentioned that the previous version of their review2 has a protocol available upon request. However, their first paper,2 which is in a Brazilian University Magazine printed in Portuguese language, does not support the updated version of the review properly since their first version does not consider any protocol in the text.\n\nTheir critical appraisal using AMSTAR-2 appears in Table 2. Five out of the 16 included review studies in their review of reviews, had between one to four AMSTAR-2 items referred as “Authors reported different information by e-mail however, it was not found in the article.” This reporting method may not be the most scholarly, safe, and respectful to present their findings, especially when the authors of their included review studies kindly accepted to provide further clarification about their methodology.\n\nParticularly, I am an author of Afrashtehfar et al.3 “Failure rate of single-unit restorations on posterior vital teeth: a systematic review,” and I regretted the online communication with their corresponding author when I was requested to provide further information. Perhaps Sarkis-Onofre et al. should have dedicated more time to conduct an adequate assessment.4 For example, their negative categorization of the AMSTAR-2 items 4, 7, and 16 for Afrashtehfar et al.3 may be mistaken. A comprehensive literature search (item 4) can be considered in the former paper3 since it searched for published papers for over 20 years with no language restriction, using four databases and displaying each search strategy in the Appendix section. Additionally, the review screened eight journals and also searched manually in the reference list of all identified relevant studies.3 Next, the list of excluded primary studies and justifications (item 7) were provided in Supplemental Table 6 (i.e., full-text excluded articles and reasons for exclusion). Regarding any potential sources of conflict (item 16), it is well-stated on the first page of the review that this study was “Supported in part by a Knowledge Transfer Grant from the Network for Oral and Bone Health Research.” Additionally, there is a section at the end of the paper for Acknowledgements where librarians and statisticians were thanked for their services.3\n\nMoreover, the search and eligibility criteria for Sarkis-Onofre et al.1 were systematic reviews that met PRISMA-P, including adults over 18 years of age with direct composite resin restoration in posterior teeth compared with other materials or techniques used in posterior teeth regardless of the outcome up to October 15th, 2017. However, some articles that fully suited their inclusion criteria were not included. For example, Afrashtehfar et al.5 “Failure of single-unit restorations on root filled posterior teeth: a systematic review” was not included despite being available from November 21st, 2016. Therefore, their search strategy and their search conduct (including the elimination of duplicates),6 as well as screening,7 raise some severe methodological concerns.8\n\nThis review of reviews has a collaboration with well-known evidence-based medicine experts from Canada, Tricco and Moher,9 which usually rely on the talent from their research team for screening and assessing the literature.\n\nAfter a brief analysis, this letter encourages the improvement of future methodological quality assessment manuscripts to:\n\n▪ Address clarification with authors of potentially included studies safely and respectfully to avoid accusation, especially if there is no consensus on the matter from different experts (i.e., two experts as a minimum).\n\n▪ Take the time and effort necessary to assess the review paper of interventions according to AMSTAR-2.4 At least two experts in the field should also determine this instead of two research students.\n\n▪ Spend sufficient time with expert librarians to develop an adequate search strategy in multiple databases.\n\n▪ Use a reference manager and do not rely on removing the duplicates by selecting only one category (i.e., authors' names). Thus, the available categories should be combined to avoid removing records that may share the same publication journal, year, or authors.\n\n▪ A PRISMA checklist10,11 should be submitted, reporting compliance with each item by indicating the paragraph and page where they can be identified in the review/appraisal. All the required reporting should be included in the quality assessment to ensure transparency and validity.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nAuthorship contribution based on the ICMJE criteria\n\nKelvin Ian Afrashtehfar: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing.", "appendix": "Acknowledgements\n\nThe author thanks Ajman University for supporting the publication of the present article.\n\n\nReferences\n\nSarkis-Onofre R, Pereira-Cenci T, Tricco AC, et al.: Systematic reviews in restorative dentistry: discussing relevant aspects. J Esthet Restor Dent. 2019. May; 31(3): 222–232. PubMed Abstract \| Publisher Full Text\n\nAquino AGR, Dias CR, Almeida FV, et al.: Características de reporte e condução de revisões sistemáticas que avaliaram a longevidade de restaurações de resina composta em dentes posteriores. Revista Da Faculdade De Odontologia – UPF. 2017; 22(1): 25.\n\nAfrashtehfar KI, Emami E, Ahmadi M, et al.: Failure rate of single-unit restorations on posterior vital teeth: A systematic review. J Prosthet Dent. 2017; 117(3): 345–353. e348. PubMed Abstract \| Publisher Full Text\n\nShea BJ, Reeves BC, Wells G, et al.: AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. BMJ. 2017; 358: j4008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfrashtehfar KI, Ahmadi M, Emami E, et al.: Failure of single-unit restorations on root filled posterior teeth: a systematic review. Int Endod J. 2017; 50(10): 951–966. PubMed Abstract \| Publisher Full Text\n\nKwon Y, Lemieux M, McTavish J, et al.: Identifying and removing duplicate records from systematic review searches. J Med Libr Assoc. 2015; 103(4): 184–188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGowan J, Sampson M: Systematic reviews need systematic searchers. J Med Libr Assoc. 2005; 93(1): 74–80. PubMed Abstract \| Free Full Text\n\nPussegoda K, Turner L, Garritty C, et al.: Systematic review adherence to methodological or reporting quality. Syst Rev. 2017; 6(1): 131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Tricco AC: Issues related to the conduct of systematic reviews: a focus on the nutrition field. Am J Clin Nutr. 2008; 88(5): 1191–1199. PubMed Abstract \| 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. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med. 2009; 6(7): e1000100. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "86654", "date": "21 Jul 2021", "name": "Marta Roqué-Figuls", "expertise": [ "Reviewer Expertise I am a statistician", "author of several systematic reviews", "and an editor in two Cochrane Review Groups", "conducting peer reviews frequently." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 comment from Afrashtehfar refers to an overview by Sarkis-Onofre et al., 20191, published in Esthet Restor Dent, which discusses key aspects of systematic reviews in restorative dentistry, focusing on the improvement of the conduct and reporting of these reviews. The author of the comment identifies specific shortcomings of the Sarkis-Onofre et al. overview and formulates some suggestions to improve future methodological quality assessment manuscripts.\nAs a general thought, this comment would surely be better suited to be published in the original journal, in order to reach the same audience that read the original paper.\nThe author of this comment is also an author of a review included in Sarkis-Onofre et al. and illustrates his criticisms of their paper with the assessments made to his review. Given this clear conflict of interest, he should be careful in formulating subjective assessments such as “Perhaps Sarkis-Onofre et al. should have dedicated more time to conduct an adequate assessment“, or using the terms ‘safe’ or ‘respectful’.\nThe author criticises the methods applied by Sarkis-Onofre et al., particularly that a single researcher conducted the study selection and AMSTAR-2 assessment. These criticisms are based on perceived errors in the AMSTAR-2 assessments made to his own review included in Sarkis-Onofre et al., and on the failure to identify and include in the overview another work he authored (Afrashtehfar et al., 20162). While these are undeniable facts and the methods applied in the overview were subpar, it’s not clear whether the errors identified are the exception or the norm, nor their impact on the overview conclusions.\nHe also points out the need for more transparency in the reporting of AMSTAR-2 assessments whenever these challenge personal communications with the original study authors. I fully support the idea to present in an appendix the clarifications about methodology provided in personal communications and the reasons for Sarkis-Onofre et al. to accept or disregard them in each case.\nHowever, there are two instances of unjustified statements made in the comment. First, the author challenges the justification of Sarkis-Onofre et al. for not registering their methodological overview in PROSPERO, arguing that as an overview it could have been registered. However, this fails to acknowledge that methodological overviews that provide no data on patient-relevant outcomes don’t verify PROSPERO registration criteria for methodological reviews.\nSecond, the author assumes that the overview, which updates a previously published overview from different authors, should follow the original overview protocol. But, while any review must have a pre-specified protocol, it's not necessary for an update to follow the protocol of the original review, as that protocol may be inexistent or outdated. What is important is that Sarkis-Onofre et al. do have a protocol available at request.\n\nThe author finishes his comment by proposing a list of suggestions for improving future methodological quality assessment manuscripts, mostly already known and common sense. However, the recommendation that AMSTAR-2 be applied by experts in the field rather than research students lacks evidence and is quite debatable.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Partly\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Partly\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "7460", "date": "24 Nov 2021", "name": "Kelvin Afrashtehfar", "role": "Author Response", "response": "I thank Prof. Roqué-Figuls for supporting the idea of presenting an appendix with methodological clarifications provided in the e-mail exchange between the overview authors (Sarkis-Onofre R, Pereira-Cenci T, Tricco AC, Demarco FF, Moher D, Cenci MS. Systematic reviews in restorative dentistry: discussing relevant aspects. J Esthet Restor Dent. 2019 May;31(3):222-232) and the comment author. Regarding the overview protocol registration, AMSTAR-2 considers “protocol registered before start of review” as a critical domain. Please refer to Shea BJ, Reeves BC, Wells G, Thuku M, Hamel C, Moran J, Moher D, Tugwell P, Welch V, Kristjansson E, Henry DA. AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. BMJ 2017; 358: j4008. Thus, one would expect that an overview of reviews (or systematic review of systematic reviews) should avoid missing such critical domain that they have assessed in their study where they used systematic reviews as the analytic unit. Indeed, the author does defend the statement that experienced researchers ideally should apply AMSTAR2 instead of inexperienced research students. Prof. Roqué-Figuls is thanked for expressing her opinions. However, the comment author did not realize of the reviewer's previous participation in the following publications: Sáenz Calvo A, Fernández Esteban I, Mataix Sanjuán A, Ausejo Segura M, Roqué M, Moher D. [Metformin for type-2 diabetes mellitus. Systematic review and meta-analysis]. Aten Primaria. 2005 Sep 15;36(4):183-91. Saenz A, Fernandez-Esteban I, Mataix A, Ausejo M, Roque M, Moher D. Metformin monotherapy for type 2 diabetes mellitus. Cochrane Database Syst Rev. 2005 Jul 20;(3):CD002966. The author accepts being concerned and kindly requests the reviewer to reconsider her decision about this comment. The comment author has complied with answering the initial reviewer's comments. Lastly, after this participation, the author has no more comments or replies." } ] }, { "id": "92392", "date": "15 Oct 2021", "name": "Luca Testarelli", "expertise": [ "Reviewer Expertise Oral Surgery", "Periodontics", "Endodontics", "Restorative Dentistry", "CBCT", "Imaging in Dentistry." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe evaluations reported in this article are of high quality and demonstrate a great knowledge of the literature and of the analysis of articles present in this article thanks to the reporting items and quality assessments.\nThe author underlines in this commentary his criticisms of the review under consideration and makes suggestions for improving the methodology and reporting of this kind of research. The author appears to be involved in Sarkis-Onofre's research, and this limits his field of action in contesting the execution of the study, underlining how another research of his has been excluded. However, we believe that the criticisms raised go beyond the conflict of interest and can be considered valid.\nAs described by the author, we agree with the need for more transparency on the methodology of these studies.\nRegarding the registration on PROSPERO, we believe that this kind of revision remains valid regardless of this. The contestation of the protocol used by the author, rather than leading to the use of standardized protocols, we believe is necessary that it be well described in the article and that it be available upon request by the authors of the study.\nThe criticisms that are posed in the article, we believe are valid and well structured. The bibliography that underlies them is thick and published in high-level journals. We, therefore, consider this article suitable for indexing, sure of the fact that it can be an added value for the quality of this type of research.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "7459", "date": "24 Nov 2021", "name": "Kelvin Afrashtehfar", "role": "Author Response", "response": "The experienced clinician-scientists and reviewers, Prof. Luca Testarelli and Prof. Rodolfo Reda, are thanked for finding value and expertise in this correspondence comment. The reviewers acknowledged that the criticisms found in this work do not represent a conflict of interest; thus, they were considered valid. Interestingly, there was also an agreement between the reviewers and the author for improved transparency on the conduction and reporting of overview studies. The reviewers found additional value in having overviews pursuing a formal repository registry and presenting the identification number. Lastly, the reviewers are acknowledged for comprehensively appraising this work and supporting the publication of this manuscript." } ] }, { "id": "93586", "date": "16 Nov 2021", "name": "Spyridon Papageorgiou", "expertise": [ "Reviewer Expertise orthodontics", "evidence-based dentisty", "systematic reviews" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with interest the abovementioned submission. However, I must say that I am somewhat confused since the author used this piece to comment on inadequacies, as deemed by the author, of a JERD-published overview of reviews that also included one of his SRs. In essence, this piece is not thought of as a standalone paper but is rather aimed at the already published overview like a correspondence with points to be addressed by the authors of the original overview in JERD. Therefore, I believe this would be more appropriate to be submitted as such in JERD and not here (not to mention that it is significantly more probable that it will be read and will be replied upon by the overview authors). Thanks for letting me see this.\nSpecific comments\nAbstract. “accessing” is probably “assessing”.\n\nBear in mind that methodological quality is different from the risk of bias. I’m not sure which of the two you are implying here, but it makes sense that it is the latter.\n\n“The improvement of future methodological quality assessment manuscripts”: this needs to be reformulated more succinctly.\n\n“Thus, this article addresses a methodological quality assessment about systematic reviews in restorative dentistry by Sarkis-Onofre et al. in the May 2019 issue of the Journal of Esthetic and Restorative Dentistry as an example of evaluating appraisals of reviews for increasing the awareness of reviewers, authors, and readers.” Instead of relaying what this piece does, better relay its’ scope/aim and its final recommendations.\n\nText. The cited paper is neither “methodological quality assessment of systematic reviews” (methodological quality assessment is a procedure) nor a “systematic review of systematic reviews”—a systematic review addresses a clinically-based research question, ideally based on PICO. The cited paper is an overview of systematic reviews.\n\nThe point about lack of registration in PROSPERO is a valid one, according to Sarkis-Onofre et al., since it complies with the instructions of PROSPERO. However, all studies that have an a priori protocol can register it (and benefit from this) and many different appropriate repositories exist.\n\nI’m not sure what is the point about the protocol being mentioned but not given. It doesn’t matter what the authors say: if there is no publicly available registered/time-stamped protocol, there is none.\n\nLikewise, I’m not sure what is meant with the 3rd paragraph. Information provided through e-mails might not necessarily be regarding the same way as published information, but can still be used in a scientific publication, if deemed appropriate.\n\nThe paragraph about the Sarkis-Onofre et al. authors wrongly judging some AMSTAR 2 fields of your SR seem to be valid, however.\n\nLikewise, the comment about potentially missed SRs to include seems to be valid.\n\nThe last bullet point might not necessarily be correct, since PRISMA is meant for systematic review—it is not a reporting guideline for overviews of review (though some fields of it might be adopted with modifications).\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Partly\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? No\n\nIs the conclusion balanced and justified on the basis of the presented arguments? No", "responses": [ { "c_id": "7458", "date": "24 Nov 2021", "name": "Kelvin Afrashtehfar", "role": "Author Response", "response": "Dr. Papageorgiou is thanked for his contribution after the correspondence author suggested him as a reviewer. The correspondence author is glad to answer his comments and clear any confusion Dr. Papageorgiou has referred to. The overview authors will be invited to reply to the correspondence author comment upon completion. As the reviewer may know, some dental journals do not accept letters to the Editor. JERD does not accept replica or letters to the Editor, whereas F1000Research provides the opportunity to have this correspondence open to the public free of charge. Thus, the latter is a different option and could be considered, by some researchers, an ideal journal to allocate the correspondence comment. Reply to the main specific comments: The correspondence author agrees with Dr. Papageorgiou that methodological quality assessment is not the same as the risk of bias. However, the correspondence author must say that an “overview of systematic reviews” is the same as a “systematic review of systematic reviews.” Kindly refer to Pollock M, Fernandes RM, Becker LA, Pieper D, Hartling L. Chapter V: Overviews of Reviews. In: Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, Welch VA (editors). Cochrane Handbook for Systematic Reviews of Interventions version 6.2 (updated February 2021). Cochrane, 2021. Available from www.training.cochrane.org/handbook. The correspondence author does understand that PROSPERO is probably the most popular repository among many other available options. Indeed, the correspondence author did refer to PROSPERO as the cited paper did. In fact, the correspondence author quoted their statement. The correspondence author appreciates that Dr. Papageorgiou found valid the point concerning the lack of PROSPERO registration. The point of mentioning PROSPERO is supported by AMSTAR-2, which considers “protocol registered before start of review” as a critical domain. Please refer to Shea BJ, Reeves BC, Wells G, Thuku M, Hamel C, Moran J, Moher D, Tugwell P, Welch V, Kristjansson E, Henry DA. AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. BMJ 2017; 358: j4008. Thus, one would expect that an overview of reviews (or systematic review of systematic reviews) should avoid missing such critical domain that they have assessed in their study where they used systematic reviews as the analytic unit. The correspondence author thanks Dr. Papageorgiou for all the other comments that he found valid. For instance, the overview is missing to include SRs, review authors wrongly judging some AMSTAR-2 fields of a previous SR by the correspondence author, and lack of repository registry. Lastly, the author appreciates Dr. Papageorgiou's comments regarding this correspondence comment. The points that could be perceived as a disagreement have been addressed by the correspondence author here." } ] } ]	1	https://f1000research.com/articles/10-442
https://f1000research.com/articles/11-220/v1	23 Feb 22	{ "type": "Systematic Review", "title": "Coffee by-products as the source of antioxidants: a systematic review", "authors": [ "Wahyu Lestari", "Kartini Hasballah", "M. Yulianto Listiawan", "Sofia Sofia", "Wahyu Lestari", "M. Yulianto Listiawan", "Sofia Sofia" ], "abstract": "Background: Solid waste from coffee depulping process threatens the organism in environment as it produces organic pollutants. Evidence suggested that coffee by-product could valorize owing to its potential as antioxidant sources. The aim of this systematic review was to evaluate antioxidant activity of coffee by-products obtained from different coffee variants (arabica and robusta) and processing methods. Methods: The systematic review was conducted as of May 29, 2021 for records published within the last ten years (2011–2021) using seven databases: Embase, Medline, BMJ, Web of Science, Science Direct, Cochrane, and PubMed. Data on type of specimen, processing methods, and antioxidant activities were collected based on PRISMA guidelines. Results: Our data suggested that aqueous extract was found to be the most common processing method used to obtain the antioxidant from various coffee by-products, followed by methanol and ethanol extract. A variety of antioxidant properties ranging from strong to low activity was found depending on the variety, type of coffee by-products (cascara, pulp, husk, silverskin, and parchment), and processing technique. Fermentation employing proper bacteria was found effective in improving the yield of bioactive compounds resulting in higher antioxidant capacity. Applications in feedstuffs, foods, beverages, and topical formulation are among the potential utilization of coffee by-products. Conclusion: Coffee by-products contain bioactive compounds possessing antioxidant properties which could be used as additives in foods, beverages, and cosmetics. In particular, their benefits in skin care products require further investigation.", "keywords": [ "robusta", "arabica", "husk", "pulp", "silverskin", "cascara" ], "content": "Introduction\n\nAs the world widely popular beverage,1,2 coffee has been produced in a large scale causing the emergence of massive organic solid waste.3,4 Most of the solid waste is originated from the depulping process, where the coffee bean is separated from the other parts of the coffee cherry.5,6 Solid waste from cherry pulp was not well-managed leading to the threat of environmental pollution.7 Nevertheless, the solid waste can be utilized for multiple purposes such as bioethanol,8 biogas,9 compost,10 and feedstuffs.11,12 Coffee by-products consist of cascara, coffee pulp, coffee husk, coffee silverskin, and coffee parchment. Among them, coffee pulp occupies the most part of coffee by-products containing carbohydrate (50%), protein (10%), fiber (20%), fat (2.5%), caffeine (1.3%), and phenolic compounds.13 The phenolic acids in cherry pulp can be detailed as followed; hydro-benzoic acid, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, gallic acid, vanillic acid, and cumaric acid.12,14 Many studies have been conducted in coffee.15–20\n\nIn dry processing, the solid waste majorly produced is husk.6 Coffee husk is rich in carbohydrate (8–85%), followed by protein (8–11%), fat (0.5–3%), and minerals (3–7%).21 Cascara obtained from husk or pulp contains natural antioxidants namely polyphenols, anthocyanin, and vitamin C along with other bioactive compounds of caffeine, alkaloids, and tannins.22 Husk is potential for human consumption due to its nature of free gluten which does not cause allergic reaction.21\n\nTaken together, coffee by-products hold a significant potential to be utilized as additives in food products.23,25 In fact, foods and beverages derived from coffee by-product have been introduced and recorded in scientific report a long time ago.12 In dermatology, the antioxidant properties from coffee by-products could provide skin protection against UV light-induced damages.26,27 Moreover, the content of polyphenols could be used for patient with alopecia, acne vulgaris, fungal infection, hyperpigmentation, or skin aging.28 Valorization of coffee by-products in a wide array of fields could offer a solution to the emerging environmental threat due to the overwhelming production of coffee solid waste.24,29\n\nSo far, the review of coffee by-products only presents the end products in general.10,24,30 Most of the reviews discussed about the application of coffee by-products in food and beverage products.12,31 Review of coffee-by products with specific topics such as topical formulation32 and polymer technology5 have been reported. Herein, we discuss the advances of coffee by-products antioxidant activities which were obtained from different coffee variants (arabica and robusta) and processing methods. To the best of our knowledge, the systematic review of antioxidant activities yielded by coffee by-products has never been published.\n\n\nMethods\n\nThis systematic review was conducted in accordance to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines33 as previously used elsewhere.34,35 Articles were included in this review, when the following criteria were fulfilled: 1). The sample was at least coffee husk, coffee silverskin, coffee pulp, coffee parchment, or the cascara (pulp and outer skin); 2). Investigated in-vitro or in-vivo anti-oxidant activities using standardized methods, as reported previously36; and 3). Published in the last 10 years (2011–2021) and written in English or Indonesian Language. Studies that only determined total phenolic compounds were not included. Editorials, reviews, commentaries, case reports, book or book chapter were excluded.\n\nThe search was conducted in May 29, 2021 through search engines of the following databases: Embase, Medline, BMJ, Web of Science, Science Direct, Cochrane, and PubMed. The terms combination used to search in the title, abstract, and keywords was “((cascara coffee) OR (coffee husk) OR (coffee pulp) OR (coffee waste)) AND ((antioxidant) OR (photoaging))”.\n\nA reference manager (EndNote X9, Thompson Reuters, Philadelphia, PA, USA) was used to import the list of references from all databases, where duplicates were then removed. The two-steps selection was carried out by firstly remove the non-eligible article by screening the titles and abstract from the collected references. Secondly, two authors WL and SS conducted the screening of the full texts according to the stated inclusion criteria and data availability. The data were extracted from main articles and their supplementary materials, whenever required. The extracted data included the report characteristics (author/s, publication year), type of specimen (coffee husk, coffee silverskin, coffee pulp, coffee parchment or cascara), processing methods (pre-treatment and extraction), and outcomes (antioxidant activity and others).\n\n\nResults\n\nThe search yielded 850 records from the stated databases (Figure 1), where as many as 170 duplicates were removed. The duplicate removal left 680 articles to undergo the first screening, of which, 616 studies were potentially eligible. Second screening excluded 597 articles, resulting the final 19 articles for qualitative synthesis.\n\nMost of the studies produced crude extract using water,22,37,44 followed by extraction using polar extracts (such as methanol and ethanol)38,40,45,46 and non-polar extract (n-hexane).47 A study, in particular, used supercritical fluid extraction with CO2 as the solvent.48\n\nThe investigations of antioxidant activities were varied in each study, but mostly, DPPH assay was employed (Table 1).6,22,40,44–46,48,52 Based on DPPH assay, the IC50 reached as low as 5.8 ppm obtained in the lotion product made of coffee pulp extract using ethanol and water solvent.46 However, the value is hard to compare since other studies did not report the IC50 and used different methods. Other antioxidant assays included FRAP,37,38,42,43 ABTS,38,43,45,49,53 NO,45,47 ORAC42,51 and Folin-Ciocalteu.45 Pre-treatments such as fermentation,47,50 vacuum drying,52 sun-drying,37 and lipophilization42,49 were found to yield the optimum value of antioxidant capacity. Other than crude extract, the reports also investigated the antioxidant properties of products made by coffee cascara such as dietary fiber,53 instant beverage powder43 lotion,46 fodder,37 anthocyanin,44 and essential oil.6,51\n\n* The most optimal value from the obtained product.\n\nDetermined by\n\na 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay;\n\nb ferric reducing antioxidant power (FRAP) assay;\n\nc 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay;\n\nd nitric oxide (NO) assay;\n\ne oxygen radical absorbance capacity (ORAC) assay;\n\nf Folin-Ciocalteu assay.\n\n\nDiscussion\n\nTreated as a solid waste in coffee industry, coffee by product could be a threat to the environment, especially to the aquatic organisms.7 Nonetheless, they could be used as the source of antioxidant into various food and beverage products, viz tea,43,54,55 dietary fiber,56,57 food preservatives,58,59 wheat flour substitute,60 and food additives.61,62 Consumption of foods or beverages derived from coffee by-products has been associated to its health benefits owing to the rich presence of bioactive compounds. Phenolic compounds and caffein have been reported contained in the coffee pulp of arabica and robusta variants.63 Phenolic compounds in high concentrations might also be retrieved from arabica coffee silverskin.64\n\nA study comparing robusta and arabica coffee pulp with aqueous extraction revealed higher antioxidant capacity in that of arabica variant.22 However, a different result was shown by another study comparing the antioxidant activities of coffee silverskin from both variants. The study found that coffee silverskin from robusta variant has higher antioxidant activity as suggested by DPPH, ABTS, and FRAP assays.65 Higher antioxidant efficacies of robusta variant were also revealed by a study employing green coffee extract.66 Antioxidant activities could be affected not only by the variant, but also the extraction or brewing method.67 Additionally, each coffee by-product could have different levels of antioxidant activity, where coffee silverskin was revealed to have the highest value.53\n\nWater has been a common solvent used on coffee by products, as reported by many researches.22,39,43,52 This due to the fact that aqueous extraction is the most practical processing method of obtaining antioxidant compounds. Moreover, it may also attract polar and semi-polar compounds such as phenolic acids, flavonoids, and so on.68,69 Other studies combined water with methanol or ethanol which can increase the affinity of the solvent with that of semi-polar compounds.38,40,45,46 Most of these studies yielded extracts with strong antioxidants (IC50 < 50 μg/mL).40,45,46,49 Lipophilization using HCl or NaOH had been proven to yield higher amount of bioactive compounds and, as a consequence, increased the antioxidant properties. The antioxidant activity could also be improved by fermentation process using proper bacteria.47,50 Nonetheless, extraction using more sophisticated solvent, such as supercritical CO2, did not contribute to higher anti-oxidant activity (IC50 > 250 μg/mL).48\n\nSince coffee by-products contain nutrients and antioxidants, they might be used as food additives or animal food sources. Simple ensiling and sun-drying were sufficient to produce fodder with high protein and fiber possessing antioxidant capacity as high as 2.5 μmol TE-1 mL.37 Dietary fiber had been isolated from various coffee by-products of both robusta and arabica variant containing bioactive compounds and high antioxidant activity.53 Coffee by-products could also be used as food odorants and dye.6,44,51 Beverage powder made of arabica cascara was proven to contain high amount of nutrients and antioxidant activity.43 The use of coffee by-product in topical formulation, such as lotion, had been reported as well.46\n\nPhenolics, flavonoids, and tannins are among the common compounds found in aqueous extracts of coffee husks, coffee pulp, and coffee, silverskin,22,38 which are identical to coffee bean.70,71 Caffeine, gallic acid, and proanthocyanidins were also contained in the by-products,48,52 contributing to the antioxidant efficacies.72 Tannins could prevent the oxidative stress, oxidative damage, and UVB-induced matrix metalloproteinase-1.73 Coffee cascara was reported to possess 8 times higher anti-radical capacity compared to blueberry with anti-cancer and vitality booster properties.52 Other health benefits of coffee by-products could be attributed to their rich fiber, magnesium, calcium, and vitamin C, and low fat content.24 Additionally, coffee cascara was also known to contain pectin which can be used as food additive.45 The aqueous extract of arabica coffee pulp could inhibit the production of IL-8 in gastric epithelial cells.45 Antibacterial properties of coffee cascara, coffee silverskin, and coffee husk have been reported as well.6,22,49\n\nBread made of coffee husk and coffee silverskin was reported free of gluten, carrying an antioxidant, α glucosidase inhibitor, which is potential to reduce chronic diseases, oxidative stress, cholesterol level, and post prandial blood glucose level.74 Arabica coffee pulp yielded high antibacterial activity against nosocomial bacteria; Staphylococcus Epidermidis and Pseudomonas Aeruginosa.75 Fermented coffee pulp had high phenolic compounds and with pH level and total acid level.76 Phenolic compounds are useful in reducing inflammation-associated cholesterol via adipogenesis inhibition.77 Anti-cholesterol properties of coffee by-product was reported to effectively decrease the cholesterol level by inhibiting the absorbance of colonic cholesterol.75\n\n\nConclusions\n\nCoffee by-products relatively have high antioxidant activities, depending on the processing method and variant. Other than antioxidants, they are rich in fiber and nutrients making them as potential additives in multiple products. Despite their high antioxidant activity and polyphenol content, the utilization of coffee by-products in topical formulation is relatively scarce compared to that in foods or beverages. Furthermore, investigation using different assays and parameters making it difficult to compare the results from each research. Hence, we recommend using robust and uniform methods in determining the antioxidant activity of coffee by-products.\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 ‘Coffee by-products as the source of antioxidants: a systematic review’. DOI: https://doi.org/10.6084/m9.figshare.18866456.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nConflict of interest\n\nThe authors declare that they have no conflict of interest.\n\n\nEthics statement\n\nNot required.", "appendix": "Acknowledgement\n\nAuthors would like to thank Postgraduate Program, School of Medicine, Universitas Syiah Kuala in assisting the during writing processes.\n\n\nReferences\n\nZainura U, Kusnadi N, Burhanuddin B: Perilaku Kewirausahaan Petani Kopi Arabika Gayo di Kabupaten Bener Meriah Provinsi Aceh. Jurnal Penyuluhan. 2016; 12(2): 126. Publisher Full Text\n\nJuliaviani N, Sahara WR: Transmisi Harga Kopi Arabika Gayo di Provinsi Aceh. Jurnal Agribisnis Indonesia. 2017; 5(1): 39–56. Publisher Full Text\n\nMoreira MD, Melo MM, Coimbra JM, et al.: Solid coffee waste as alternative to produce carotenoids with antioxidant and antimicrobial activities. Waste Manag. 2018 Dec; 82: 93–99. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "128542", "date": "11 Apr 2022", "name": "Emil Salim", "expertise": [ "Reviewer Expertise Natural product", "innate immunity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCoffee depulping processes generate solid waste that threatens the environment. Many researchers reported that coffee by-products have the potential as antioxidant sources. This systematic review aimed to assess the antioxidant activity of coffee by-products derived from various coffee varieties (arabica and robusta) and processing techniques. As of May 29, 2021, this study was carried out for papers published within 2011-2021 using Embase, Medline, BMJ, Web of Science, Science Direct, Cochrane, and PubMed databases.\nPRISMA guidelines were used to collect information on the type of specimen, processing procedures, and antioxidant activity. Aqueous extraction was the most common method to obtain the antioxidant from various coffee by-products, followed by methanolic and ethanolic extraction. The antioxidant properties varied depending on coffee by-products (cascara, pulp, husk, silverskin, and parchment) and processing method. Coffee by-products contain antioxidant compounds that could be employed as food, beverage, and cosmetic additives.\nThis systematic review is critical for identifying, evaluating, and summarising the antioxidant activity of coffee by-products obtained from different coffee varieties (arabica and robusta) and processing procedures. The review’s rationale, objective, and method were clearly stated, and the selected reports support the conclusion.\nHowever, to improve the quality of this review, I suggest some corrections:\n“Results: Our data suggested that aqueous extract was found to be the most common processing method used to obtain the antioxidant from various coffee by-products, followed by methanol and ethanol extract.” I suggest that aqueous extract…..methanol and ethanol extract are replaced by aqueous extraction…..methanol and ethanol extraction.\n\n“Many studies have been conducted in coffee.” This sentence is unclear; please add more information about the studies.\n\n\"Phenolics, flavonoids, and tannins are among the common compounds found in aqueous extracts of coffee husks, coffee pulp, and coffee, silverskin,22,38 which are identical to coffee bean.\" It should be 'coffee silverskin' (without a comma).\n\nStaphylococcus Epidermidis and Pseudomonas Aeruginosa should be written as Staphylococcus epidermidis and Pseudomonas aeruginosa.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "136770", "date": "20 May 2022", "name": "Ghodratollah Panahi", "expertise": [ "Reviewer Expertise Diabetes", "endocrine disorder", "miRNA", "molecular Biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this research, the authors systematically reviewed the studies that evaluated the antioxidant activity of coffee by-products obtained from different coffee variants. They provide evidence that, depending on the processing method and variant, coffee by-products have high antioxidant activities. It is suggested that these compounds could be used as additives in foods, beverages, and cosmetics but their benefits in skincare products require further investigation.\nThe article is well written and methodologically well done. Congratulations to the authors of this research, and I think the article is ready to be accepted and published.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-220
https://f1000research.com/articles/11-215/v1	22 Feb 22	{ "type": "Research Article", "title": "Tolerability of COVID-19 mRNA vaccines in patients with postural tachycardia syndrome: a cross-sectional study", "authors": [ "Karin Jost", "Belén Rodriguez", "Nicole Söll", "Robert Hoepner", "Werner J. Z'Graggen", "Karin Jost", "Belén Rodriguez", "Nicole Söll", "Robert Hoepner" ], "abstract": "Background: Postural tachycardia syndrome (POTS) is a form of autonomic dysregulation. There is increasing evidence that the etiology may be immune-mediated in a subgroup of patients. Patients with POTS often experience an exacerbation of their symptoms associated with (viral) infections and often fear the same symptom aggravation after vaccination. In this report we describe the tolerability of messenger ribonucleic acid (mRNA) vaccines against coronavirus disease 19 (COVID-19) and the consequences of a COVID-19 infection on POTS symptoms in our cohort of patients with neuropathic POTS.\nMethods: We conducted a standardized, checklist-based interview with 23 patients and recorded the acute side effects of mRNA vaccination, acute symptoms of COVID-19 infection as well as the effects of vaccination and COVID-19 infection on POTS symptoms.\nResults: Of all included patients, 20 patients received two mRNA vaccines without having had a previous COVID-19 infection, and five patients in total had suffered a COVID-19 infection. Of these, three had COVID-19 without and two after being vaccinated. No increased frequency of side effects after both doses of mRNA vaccines was observed. Six patients reported a mild and short-term aggravation of their POTS symptoms beyond the duration of acute vaccine side effects. All five patients who suffered a COVID-19 infection subsequently reported a pronounced and persistent exacerbation of POTS symptoms.\nConclusions: Our observations suggest that mRNA vaccines are not associated with a higher frequency of acute side effects in patients with POTS. Symptom exacerbation as a consequence of mRNA vaccination seems to be less frequent and of shorter duration compared to patients who suffered a COVID-19 infection.", "keywords": [ "autonomic dysfunction", "orthostatic intolerance", "autoimmune", "autonomic neuropathy" ], "content": "Introduction\n\nPostural tachycardia syndrome (POTS) results from autonomic dysregulation. It is characterized in adults by a clinically symptomatic, sustained increase in heart rate of more than 30 beats per minute within 10 minutes of standing or head-up tilt testing, in the absence of orthostatic hypotension.1,2 Patients with POTS experience symptoms of orthostatic intolerance in the upright position such as lightheadedness, dizziness, palpitations, tremulousness, generalized weakness and leg pain, blurred vision, dyspnea, nausea, headache and cognitive dysfunction.3–6 Many patients with POTS additionally report non-orthostatic symptoms of autonomic origin such as fatigue, gastrointestinal complaints, sleep disturbances, restless legs symptoms and exercise intolerance.7,8 The exact etiology of POTS is still unknown, although in recent years evidence has accumulated that in a subset of patients with POTS the pathogenesis of dysautonomia may be immune-mediated.9,10 The onset of POTS is frequently reported after an immunologic stressor, with a female predominance.11,12 Up to 50% of patients with POTS describe a viral infection as the trigger of their symptoms.12,13 Patients also often report that infections (especially viral illnesses) are triggers for a prolonged symptom exacerbation, even after the subsiding of the acute infection.4,13,14 Individuals with POTS are more likely to be affected by comorbid autoimmune diseases than the average population.8,15,16 In recent years, autoantibodies against G-coupled protein receptors, most often including autoantibodies against adrenergic and cholinergic receptors, were characterized in POTS.9,17–19 Autoantibodies against the α1-adrenergic receptor were the most common among them.18–20 Additionally, antibodies against angiotensin II type 1 receptors and abnormal levels of inflammatory biomarkers were reported.21,22 Despite the presence of these antibodies, their role in the complex pathophysiology of autonomic dysfunction in POTS remains unknown.15 Immunomodulatory treatment with intravenous immunoglobulins has shown a positive effect on the symptoms of patients with POTS, further supporting an immune-mediated genesis.23\n\nIn the wake of the coronavirus disease 2019 (COVID-19) pandemic, numerous case reports and case series about the occurrence of POTS following an infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have accumulated.24–30 Nearly all affected individuals were females without pre-existing conditions who developed symptoms of autonomic dysfunction several days or weeks after an acute COVID-19 infection and there was no association with initial COVID-19 severity.24,31,32 Vaccines based on messenger ribonucleic acid (mRNA) technology are being used to combat the COVID-19 pandemic. The mRNA provides the body with the genetic code of the virus, which is then translated in the host cells and as a consequence, spike proteins are built. These act as antigens and trigger an immune response, as a result of which neutralizing antibodies against SARS-CoV-2 are formed.33 There is one case report in which POTS was diagnosed in a previously healthy, 42-year-old male following the first dose of mRNA vaccination.34\n\nWe have observed that patients with POTS are hesitant towards vaccination in general and especially towards the new mRNA vaccines because they often fear aggravation of their symptoms. On the other side, it is reasonable to assume that a COVID-19 infection in patients with POTS may trigger a prolonged symptom amplification as it is commonly observed with infections.\n\nThe aim of this study was to assess the tolerability and side effects of the two COVID-19 mRNA vaccines used in Switzerland (Spikevax®, Moderna; BNT162b2®, Pfizer) in a cohort of patients with POTS, and to assess possible consequences of a COVID-19 infection on POTS symptoms.\n\n\nMethods\n\nWe conducted a standardized checklist-based interview with all patients who had been diagnosed with neuropathic POTS and were followed in the Autonomic Unit of the Departments of Neurology and Neurosurgery, University Hospital Bern, Bern, Switzerland. All available patients were contacted by telephone and asked if they were interested in participating in the study after checking the eligibility criteria. If interested, they were sent the informed consent form. After receiving the signed consent form the interview took place. The structured interviews were performed by one of two authors (KJ or BR) either by telephone or during a routine consultation. Data was collected between November 2021 and January 2022. All contacted patients agreed to participate in the study and provided written informed consent for the collection and publication of their data. Potential bias was minimized by the standardization and structuring of the interview. The interviewer strictly followed the predetermined interview checklist (please see the extended data for the used interview checklist).48 The study was carried out in accordance with the Declaration of Helsinki. The diagnosis of POTS had been made according to medical history, physical and neurological examination, cardiovascular autonomic function testing, thermoregulatory sweat test and/or quantitative testing of sudomotor axon reflex, determination of autoantibodies against G-protein-coupled receptors, measurement of plasma norepinephrine levels and skin biopsy in selected patients.1,35\n\nPatients had to meet the following inclusion criteria: confirmed diagnosis of neuropathic POTS, aged between 18 and 60 years, received two COVID-19 mRNA vaccine doses ≥ 1 month prior to the interview, or recovered from COVID-19 infection ≥ 1 month prior to the interview.\n\nTo evaluate the tolerability of COVID-19 mRNA vaccines, the following data were collected during the interviews: Date(s) of vaccination and type of vaccine (BNT162b2®, Pfizer BioNTech, New York, NY or Spikevax®, Moderna, Cambridge, MA). The following, previously published side effects of vaccines36–38 were assessed (for the first and second dose of the vaccine separately) in their presence (yes/no) and duration (days): fever, shivering, fatigue, headache, joint pain, muscle pain, nausea, emesis, diarrhea, and reaction at injection site (pain, swelling and cutaneous reaction).49\n\nIn patients who had suffered a COVID-19 infection, the following additional symptoms were queried: coughing, sore throat, rhinorrhea, breathlessness, loss of taste, loss of smell and chest pain. For each symptom, the presence (yes/no), severity (mild, moderate, severe) and duration (in days) were evaluated. Furthermore, the duration of the infection, need for hospitalization and incapacity for work were assessed.\n\nTo assess possible exacerbation of POTS symptoms due to mRNA vaccination and COVID-19 infection, the presence (yes/no), severity (mild, moderate, severe; for COVID-19 infection only) and duration of symptom exacerbation (in days) for the following symptoms were evaluated: dizziness, nausea, weakness, palpitations, lightheadedness, tremulousness, blurred vision, concentration difficulties, memory difficulties, orthostatic leg and/or arm pain, gastrointestinal symptoms, sleep disturbances, restless legs syndrome and orthostatic headache. During the interview, symptom aggravation was assessed separately for the first and second dose of the vaccine, and COVID-19 infection, from the patients memory. Furthermore, adjustment of therapy and inability for work due to symptom exacerbation were assessed.\n\nThe data analysis was descriptive and performed using Statistical Package for the Social Sciences (SPSS Statistics) Version 25.0 (IBM Corp., Armonk, NY, USA). Data are reported either as frequencies, mean (range) or median (range). All interviews were fully completed, so there were no missing data.\n\n\nResults\n\nA total of 23 patients, two men (8.7%) and 21 women (91.3 %) with diagnosed neuropathic POTS and a mean age of 26.65 (range 18-40) years, were included in this study and interviewed once. In total, 20 patients who had been vaccinated twice and had not previously suffered a COVID-19 infection were assessed for side effects of the vaccinations. Of the 23 patients included in this study five (21.7%) had suffered a COVID-19 infection; three before and two after two doses of mRNA vaccination.48\n\nFrequencies of published acute side effects of mRNA vaccination reported by our POTS cohort are shown in Table 1. All included patients received the first dose between April and September 2021 and the second dose between May and October 2021. After the first dose, patients were unable to work for a mean of 0.35 (range 0-7) days and after the second dose for a mean of 1.05 (range 0-3) days. No allergic reactions were observed.\n\nAcute symptoms of COVID-19 infection are summarized in Table 2. Mean duration of infection was 16.4 (range 10 – 27) days. None of the patients had to be hospitalized. Mean duration of incapacity for work was 18.8 (range 10 – 28) days.\n\n* COVID-19 infection after 2 doses of mRNA vaccine (Spikevax®, Moderna).\n\nReported increase of POTS symptoms after mRNA vaccination is shown in Table 3. An increase of POTS symptoms was reported by three patients after the first and by five patients after the second vaccination. Mean duration of symptom increase was seven days (range 1-14). None of the patients needed an adjustment of the symptomatic therapy for POTS, and no incapacity for work was reported.\n\nThe effects of COVID-19 infection on POTS symptoms are shown in Table 4. In addition to the above reported incapacity for work, one patient (Patient 3) had to reduce her existing workload for two more months. Adjustment of symptomatic POTS treatment was necessary in all patients.\n\n* COVID-19 infection after 2 doses of mRNA vaccine (Spikevax®, Moderna).\n\n\nDiscussion\n\nThe present study investigated the frequencies of known side effects of mRNA vaccination (Spikevax®, Moderna; BNT162b2®, Pfizer) in patients with POTS. In addition, possible effects on POTS symptoms were assessed and compared to the impact of a COVID-19 infection.\n\nVaccine side effects were present in 20 (100%) patients for both vaccinations. The most frequently reported side effects of mRNA vaccines were pain at the injection site (70% after first, 85% after second), fatigue (50% after first, 80% after second), headache (30% after first, 75% after second), fever (20% after first, 65% after second) and shivering (15% after first, 65% after second). Side effects were generally reported more frequently after the second vaccination. This is in line with the results of other studies investigating the side effects of mRNA vaccines on healthy subjects as well as with the data from the vaccine manufacturers.33,36–38\n\nOnly six patients reported mild worsening of their POTS symptoms after vaccination beyond the duration of the acute side effects, for a mean duration of seven days (maximum 14 days). The observed increase in symptoms occurred more frequently after the second vaccination. This is similar to findings of studies examining the effects of mRNA vaccination on disease activity in patients with autoimmune inflammatory rheumatic diseases, which showed no higher incidence of side effects compared to healthy subjects and no greater risk of disease flares.39,40 Similarly, also patients who suffered from post-COVID symptoms of dysautonomia did not report a worsening of symptoms after getting vaccinated.41\n\nIn contrast, patients suffering a COVID-19 infection experienced a pronounced and prolonged aggravation of their POTS symptoms for several months. Due to the symptom increase all patients needed an adjustment of their symptomatic POTS therapy and had prolonged incapability for work. Interestingly, symptom exacerbation due to a COVID-19 infection was also observed in two previously vaccinated patients. However, both patients were vaccinated more than six months prior to the infection at a time when booster vaccinations were not yet available for this priority group in Switzerland. In these two patients, there was a tendency for a milder and shorter exacerbation of POTS symptoms compared to non-vaccinated patients.\n\nMost patients with POTS experience a prolonged increase of their symptoms in the context of infections (especially of viral etiology).4,13,14 In general, hypovolemia, fever and bedrest can intensify POTS symptoms.26,42,43 Furthermore, in patients with possible immune-mediated POTS, symptom aggravation is most likely due to a general immunological activation. Besides this, SARS-CoV-2 appears to affect the autonomic nervous system directly, which could be an additional factor for aggravation.44 Since the onset of the COVID-19 pandemic, an increasing number of case reports about the occurrence of POTS secondary to a COVID-19 infection have emerged.24–30 Several hypotheses about possible pathomechanisms of POTS or dysautonomia in general after COVID-19 infection have been proposed: imbalance of the renin-angiotensin-aldosterone system,26,29 brainstem involvement,43,45,46 autoreactivity to antibodies against SARS-CoV-2,25,43,47 dyshomeostasis of immune response42,44 and denervation of peripheral sympathetic nerve fibers. 26,43,42\n\nThis study has some limitations. Due to the small number of cases (especially of POTS patients with COVID-19 infection), generalizability cannot be fully derived. Furthermore, the retrospective collection of data by interview bears the risk of inaccurate symptom recollection and reporting in patients. Finally, effects of mRNA booster vaccinations and other types of vaccination were not recorded in this study.\n\n\nConclusion\n\nThe observations of this study suggest that mRNA vaccines are not associated with a higher incidence of acute side effects in patients with POTS and only pose a mild to moderate risk for POTS symptom exacerbation, usually of short duration. POTS symptom exacerbation as a consequence of mRNA vaccination was milder and of shorter duration compared to patients who suffered a COVID-19 infection.\n\n\nEthical statements\n\nThis study was carried out in accordance with the recommendations of the local ethics committee (Kantonale Ethikkommission Bern, Switzerland, project-ID: 2021-02115; 02.11.2021).\n\nAll subjects gave written informed consent for publication of these data in accordance with the Declaration of Helsinki.\n\n\nData availability\n\nDryad: Tolerability of COVID-19 mRNA vaccines in patients with postural tachycardia syndrome https://doi.org/10.5061/dryad.zkh1893bx.48\n\nThis project contains the following underlying data:\n\n- Demographic_data.xlsx\n\n- POTS_symptoms_after_COVID-19.xlsx\n\n- POTS_symptoms_after_vaccination.xlsx\n\n- Side_effects_of_mRNA_vaccines.xlsx\n\n- Symptoms_of_COVID-19_infection.xlsx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nZenodo: Tolerability of COVID-19 mRNA vaccines in patients with postural tachycardia syndrome https://doi.org/10.5281/zenodo.5925527.49\n\n- Informed_Consent_Form.pdf\n\n- Interview_Checklist.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nFreeman R, Wieling W, Axelrod FB, et al.: Consensus statement on the definition of orthostatic hypotension, neurally mediated syncope and the postural tachycardia syndrome. 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Publisher Full Text\n\nSheldon RS, Grubb G II, Shen W-K, et al.: 2015 Heart Rhythm Society Expert Consensus Statement on the Diagnosis and Treatment of Postural Tachycardia Syndrome, Inappropriate Sinus Tachycardia, and Vasovagal Syncope. Hear Rhythm. 2017; 12(6): e41–e63. Publisher Full Text\n\nMeo SA, Bukhari IA, Akram J, et al.: COVID-19 vaccines: Comparison of biological, pharmacological characteristics and adverse effects of pfizer/BioNTech and moderna vaccines. Eur. Rev. Med. Pharmacol. Sci. 2021; 25(3): 1663–1679.\n\nFDA-KAIRA: Fact Sheet for Healthcare Providers Administering Vaccine. US Food Drug Adm. 2020; 2019: 1–22. Reference Source\n\nHCP Fact Sheet-EUA Full PI_Pfizer-BioNTech COVID-19 vaccine_PBS 30 mcg_purple_1.3.2022 final.2022;2019(January): 1–54. 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PubMed Abstract \| Publisher Full Text\n\nGoldstein DS: The possible association between COVID-19 and postural tachycardia syndrome. Hear Rhythm. 2021; 18(4): 508–509. PubMed Abstract \| Publisher Full Text\n\nGoldstein DS: The extended autonomic system, dyshomeostasis, and COVID-19. Clin. Auton. Res. 2020; 30(4): 299–315. PubMed Abstract \| Publisher Full Text\n\nBlitshteyn S: Is postural orthostatic tachycardia syndrome (POTS) a central nervous system disorder?. J. Neurol. 2021; 269(0123456789): 725–732. PubMed Abstract \| Publisher Full Text\n\nBisaccia G, Ricci F, Recce V, et al.: Post-acute sequelae of covid-19 and cardiovascular autonomic dysfunction: What do we know?. J. Cardiovasc. Dev. Dis. 2021; 8(11): 1–15. Publisher Full Text\n\nWallukat G, Hohberger B, Wenzel K, et al.: Functional autoantibodies against G-protein coupled receptors in patients with persistent Long-COVID-19 symptoms. J. Transl. Autoimmun. 2021; 4(April): 100100–100105. Publisher Full Text\n\nJost K, et al.: Tolerability of COVID-19 mRNA vaccines in patients with postural tachycardia syndrome, Dryad. Dataset. 2022. Publisher Full Text\n\nJost K, et al.: Tolerability of COVID-19 mRNA vaccines in patients with postural tachycardia syndrome, Zenodo. Dataset. 2022. Publisher Full Text" }	[ { "id": "135527", "date": "13 May 2022", "name": "Johann Sellner", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important contribution in the wake of vaccination hesitancy among people with neurologic disease. The study provides early scientific evidence that POTS, a condition that may also occur after infections, is not associated with an increased risk of side effects of SARS-CoV-2 vaccination. Importantly, pronounced and prolonged symptom exacerbation was observed with COVID-19 in patients with POTS. This observation should serve as an additional line of argumentation for the safety and efficacy of SARS-CoV-2 vaccination and identifies patients with POTS as a vulnerable cohort for detrimental outcome of COVID19. The only remark from my side relates to the lack of a control group, ideally sex- and age-matched.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "8253", "date": "24 May 2022", "name": "Belén Rodriguez", "role": "Author Response", "response": "Thank you for your review. We agree with you that a sex- and age-matched control group would have further improved the power of the study. Due to the extensive data already available on the tolerability of the vaccine in healthy populations, we decided not to include a control group." } ] }, { "id": "209338", "date": "09 Oct 2023", "name": "Jorge Rodríguez-Capitán", "expertise": [ "Reviewer Expertise Postural tachycardia syndrome related to COVID-19 desease or related to mRNA vaccinatio" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an interesting study on a specific, well-defined topic for which there is limited prior evidence, and which also represents a real problem in a group of patients: the safety of mRNA COVID-19 vaccines in patients with postural tachycardia syndrome (POTS). Thus, in my opinion, the authors deserve congratulations for this work in which safety data are provided after vaccination in this patient group, something that is crucial to prevent the undesirable effects of a low vaccination rate in this patient population.\nHowever, the study has some limitations, most of which have been acknowledged by the authors. Firstly, it is a single-center study with a small sample size. Furthermore, a comparison is made between the worsening of symptoms related to POTS after COVID-19 and the worsening observed after vaccination. The conclusion, based on data from a small number of patients (only 5 post-COVID-19 cases), is that the worsening of symptoms after vaccination is of much lower intensity and duration than after COVID-19. In addition, it is suggested that the worsening of POTS symptoms after COVID-19 is of lower intensity and duration in previously vaccinated patients compared to unvaccinated patients, but this assertion can only be based on 3 unvaccinated patients compared to 2 vaccinated patients. This small sample size limits these conclusions, as acknowledged by the authors.\nIn any case, the data do show that vaccination was well-tolerated in patients with POTS, and although it cannot be demonstrated that COVID-19 illness after vaccination reduces the degree of worsening of POTS symptoms, the benefits in terms of preventing severe COVID-19 disease outweigh the risk/benefit balance in favor of vaccination in this patient group.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10368", "date": "10 Oct 2023", "name": "Belén Rodriguez", "role": "Author Response", "response": "Thank you for your review. We agree with you that the conclusion that exacerbation of POTS symptoms could be worse after COVID-19 infection than after mRNA vaccination is limited by the small sample size. As the topic of vaccination was very important and relevant at the time, and may become relevant again in the future, we believe that our results still could contribute to the decision-making by other clinicians in this field, despite the limited sample size." } ] } ]	1	https://f1000research.com/articles/11-215
https://f1000research.com/articles/11-212/v1	22 Feb 22	{ "type": "Research Article", "title": "COVID-19: Comparison of immunogenicity response between natural and post-vaccination infections", "authors": [ "Ivonne Elisabeth Rotty", "Erwin Kristanto", "Sekplin Sekeon", "Henny Ruth Liwe", "Neni Ekawardani", "Sekplin Sekeon", "Henny Ruth Liwe" ], "abstract": "Background: The COVID-19 pandemic in Indonesia has been ongoing for a year as at time of writing, since March 2020. Vaccination interventions are public health efforts that are arguably the most effective in the current pandemic situation, in addition to routine health protocols. Until now, there have been few reports of the effectiveness of vaccination and antibody titers formed after vaccination is carried out. This study aims to find out the difference in antibody titers after vaccination in confirmed COVID-19 cases. Methods: This observational study investigated the difference in SARS-Cov-2 quantitative antibody titers between two cohorts: unvaccinated COVID patients who were confirmed -with COVID-19 and individuals undergoing vaccination at the hospital Prof. dr. R. D. Kandou Manado. Inclusion and exclusion criteria, statistical analysis, and research ethics were applied in the study. Results: Antibody titers in survivor groups were relatively lower at 56 days and 84 days after COVID-19 diagnosis, while the antibody titer in the elderly group undergoing vaccination relatively increased at 56 days and 84 days after the first vaccination. There was a significant difference in antibody titers between a group of survivors and those who underwent vaccination on the first (28 days) and third (84 days). Conclusions: From this study, it was found that in the naturally COVID-19-infected group, antibody titers were still found for 84 days after COVID-19 diagnosis. In the group undergoing vaccination, it was found that antibody titers increased significantly at 56 days after vaccination.", "keywords": [ "COVID-19", "Immune response", "Survivor", "Vaccination", "Antibody Titer" ], "content": "Introduction\n\nThe COVID-19 pandemic in Indonesia has been running for a year since March 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) can cause respiratory failure and even multiple organ failure (Pal et al., 2020). Vaccination efforts are needed to prevent the burden of pandemics in the form of increased morbidity and mortality, and to reduce the rate of transmission of the SARS-Cov-2 virus.1\n\nVaccination interventions are arguably the most effective public health efforts in the current pandemic situation, in addition to routine health protocols (Dinleyici et al., 2021).2 The vaccine can protect individuals against SARS-Cov-2 infection, as well as suppress the severity of the COVID-19 disease (Pascual-Igleas et al., 2021). However, no vaccine provides 100% total protection.3 After vaccination, information is needed about the proportion of communities that have had an immune response after the vaccination program. Thus, the evaluation of antibodies produced after vaccination is necessary.\n\nResearch to find humoral responses in adults aged 60 years and older was conducted by Zhiwei Wu et al. in 2020.4 The study was carried out on 72 respondents with placebo and CoronaVac vaccination interventions in two separate groups and was carried out in two phases with a time interval of 28 days. The results of this study showed that CoronaVac is well tolerated and induces a humoral response in adults aged 60 years and older, which supports the use of this vaccine in older populations.\n\nUntil now, there are still few reports of the effectiveness of vaccination and antibody titers formed after vaccination is carried out. Furthermore, not many publications have reported differences in antibody production after vaccination compared to the antibody titers of unvaccinated, recovered COVID-19 patients. Furthermore, it is necessary to know the difference in the duration of the formation of natural post-infectious antibodies and post-vaccination. Therefore, this study aimed to find out the difference in antibody titers after vaccination, in individuals with confirmed COVID-19.\n\n\nMethods\n\nThe type of study conducted was a cohort observation, investigating the difference in SARS-Cov-2 quantitative antibody titers between patients who were confirmed, unvaccinated COVID-19 cases, and individuals who underwent vaccination at the hospital Prof. dr. R. D. Kandou Manado. The study was conducted at the Prof. dr. R. D. Kandou Hospital from March to August 2021.\n\nThe study population was confirmed, unvaccinated of COVID-19 patients treated in the COVID-19 infectious treatment room of Prof. dr. R. D. Kandou Hospital and individuals who underwent vaccinations at Prof. dr. R. D. Kandou Hospital.\n\nThis study consisted of two groups, naturally infected COVID-19 patient and individual who underwent vaccination, with a sample size of 30 for each group. The study subjects were samples that met the inclusion criteria described below.\n\n\n\n‐ SARS-Cov-2 quantitative antibody titer: The results of the measurement of specific antibodies with the Electro Chemiluminescence Immunoassay (ECLIA) method, using recombinant proteins representing the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein in the serum of patients, measured as U/ml units (1U/ml equivalent to 0.972 BAU/ml of international standard units from WHO).\n\nIn this study, we obtained antibody titer results using the ArchitectPlus i1000SR tool made by Abbot, USA with three types of reagents used, namely:\n\na. SARS Cov-2 immunoglobulin G (IgG) antibody reagent with catalogue number 28453FN00.\n\nb. Sars Cov-2 IgG antibody reagent control with catalogue number 3056FN00.\n\nc. Sars Cov-2 IgG calibration antibody reagent with catalogue number 29156FN00.\n\n‐ SARS-Cov-2 survivor: People who survived infection with COVID-19, or people who recovered from COVID-19.\n\n‐ Individuals undergoing vaccination: Individuals receiving an injection of the COVID-19 vaccine.\n\n‐ Confirmed patients with SARS-Cov-2: People who have tested positive for the coronavirus based on the results of laboratory examinations.\n\n\n\n‐ Aged 18 years or older.\n\n‐ Willing to participate in the research.\n\n‐ Confirmed COVID-19 patients, i.e., confirmed COVID-19 cases treated in infectious treatment rooms or in the outpatient COVID-19 polyclinic at RSUP Prof. dr. R. D. Kandou Hospital.\n\n‐ Individuals have undergone SARS-Cov-2 vaccination at Prof. dr. R. D. Kandou Hospital.\n\n‐ Willing to be contacted to conduct further checks.\n\n\n\n- Individuals who did not continue or did not get a second vaccination.\n\n- Survivors who have been vaccinated.\n\n- Confirmed COVID-19 patients who died in treatment.\n\n- Subjects who dropped out in the course of the study.\n\nConfirmed COVID-19 patients treated as confirmed COVID-19 cases and individuals undergoing vaccination were included in the study after passing the selection criteria. Most of the participants belonged to the non-elderly age group, so this group was further divided into two more groups, those with comorbid and without comorbid, with the same number of samples as elderly respondents. Sampling was carried out with a simple stratified random sampling technique for each group.\n\nAfter a period of 28, 56, and 84 days (test 1, 2 and 3 respectively) after the diagnosis of COVID-19 for confirmed COVID-19 patients, a blood sample examination was carried out to measure SARS-CoV-2 antibody titer levels. Quantitative Anti-Cov-2 examination is a test that measures quantitative in vitro levels of antibodies (including IgG) specific to the RBD of the SARS-CoV-2 S protein; it aims to assess the adaptive humoral immune response to the SARS-CoV-2 S protein.\n\nThe same measurement was done for individuals who underwent SARS-Cov-2 vaccination.\n\nComparison of SARS-Cov-2 antibody titer levels was conducted between the group of unvaccinated patients with confirmed COVID-19 and individuals undergoing vaccination.\n\nSecondary data (demographic characteristics) such as age, sex, elderly status, and infection status were taken from the patient's medical record.\n\nA descriptive analysis was carried out on the demographic and clinical criteria of the research subject.\n\nBivariate analysis with an independent T-test (if data distribution was normal) or was carried out with Mann-Whitney U test (if the data were not normally distributed) was carried out to determine the difference in the average antibody titers between the two groups. Furthermore, to assess the difference in antibody levels in all three tests for each group of study subjects, the Friedman two-way ANOVA by rank test followed by post hoc analysis with the Bonferroni method were used.\n\nThe analysis was conducted with a significance level of 5% using SPSS version 20 software.\n\nThe research protocol was reviewed according to the standards of the Council of International Organizations of Medical Sciences (CIOMS).\n\nThe subject of the study was related to the principle of good clinical practice.\n\nEthical clearance was submitted to the Health Research Ethics Committee of RSUP Prof. dr. R. D. Kandou and was issued with Number 041/KEPK-KANDOU/III/2021 The research was submitted to the President Director of RSUP Prof. dr. R.D. Kandou Manado. All subjects or participants whose data are listed in the paper have agreed by signing the Informed Consent.\n\n\nResults\n\nOf the total sample constituted of 65 people, nine subjects had incomplete results (i.e., died or discontinued participation to the study) and were therefore excluded from the analysis. The distribution of the study subjects’ demographic characteristics can be seen in Table 1, where the subjects are classified by age group, elderly status, sex, and vaccination status.\n\nBased on demographic characteristics data in study subjects, it was found that most belonged to the non-elderly age group. Female gender made up the majority with 60.7%. The proportions were balanced between the group of survivors and vaccinations. Table 2 shows that 37.5% of the subjects who participated in this study were in the elderly group and 62.5% of the subjects were in the non-elderly age group.\n\nFrom the results of the cross-table analysis above, it was found that there was a relatively balanced composition between the group of survivors and vaccinations in both the elderly and non-elderly categories.\n\nA bivariate analysis was conducted to see differences in antibody levels in the elderly and non-elderly groups, followed by an other analysis to see the difference between the status of survivors and vaccinations in each age group, both elderly and non-elderly.\n\nBased on the results of the distribution of antibody levels in the form of median values in the elderly group (Figure 1), it was observed that antibody levels in the elderly survivor group were decreased, which appeared to be a 50% reduction in the median value between the first and last test. In contrast, in the elderly group who underwent vaccination, there was a widening of the interquartile range comprising 50% of the values around the median for test 1 to test 3.\n\nWhen the Mann-Whitney U test was conducted to see the difference in antibody levels of each group in each test, it was found that there was a statistically significant difference in the results of test 1 (Table 3).\n\nIn the non-elderly group without comorbidities, the distribution of median values showed a decrease (Figure 2), as shown by lower median scores for tests 2 and 3 compared to tests 1. On the other hand, there was an increase in median scores in the vaccination group, coinciding with a widened interquartile range for values from tests 2 and 3; these values were higher and had a wider distribution compared to test 1 (Figure 2).\n\nBased on the calculation results from the Mann-Whitney U test, there was a statistically significant difference in both of the non-elderly survivors without comorbidities and those in the vaccinated group (Table 4).\n\nThe spread of antibody levels in non-elderly groups with comorbidities showed a decrease in median values for survivors. However, the pattern was also relatively similar for the group that underwent vaccinations. Visually, there was also a reduction in the spread of the interquartile range for both groups (Figure 3).\n\nThe visual patterns described above were confirmed by the absence of significant differences between the groups of survivors and vaccinated patients, based on the results of the Mann-Whitney U test (Table 5).\n\nWhen sorting the total sample according to survivor and vaccination status, ignoring the age group variable, a decrease in the median value in the survivor group appeared. There was also a decrease in the median scores of tests 2 and 3 compared to tests 1 (Figure 4). On the contrary, there was an increase in the median score in the vaccination group, with higher values for tests 2 and 3 than that of test 1.\n\nThere was a significant difference in the median antibody levels of the survivors' group and that of vaccinated subjects between tests 1 and 3. This is likely due to changes in median levels in both.\n\nFurthermore, to assess the difference in antibody levels in all three tests for each group of study subjects, the Friedman two-way ANOVA by rank test followed by post hoc analysis with the Bonferroni method were used. The goal was to analyze the differences in each test, and if there were differences, to determine which tests had significantly different values (Table 7).\n\nThe results of the analysis with the Friedman test above showed statistical differences in antibody levels in all three tests conducted, for all categories except in young age groups with comorbidities and not vaccinated (x2 = 2.00, p = 0.37).\n\n\nDiscussion\n\nIn this study, the majority of subjects belonged to the age group over 60 years, with a proportion of 32.1%, followed by the age group 41-50 years (23.2%) and 51-60 years (21.4%). However, in general, the young age group composition still dominated compared to the elderly group (62.5% versus 37.5%). This is in line with various reports of COVID-19 infection being more widely found in adult to elderly groups (Van Jaarsveld, 2020).5 Based on cross-description data, the study found a relatively balanced comparison between elderly survivors and vaccinated patients. In contrast, the younger age group was dominated by survivors, with a proportion of 32.1%.\n\nThe present study (Table 6) show that (The results of this study generally showed significant) differences in antibody titer levels in the elderly group of survivors and vaccinated individuals, especially for the first test. This difference then became insignificant at the time of the second and third tests. The elderly survivor group showed differences in antibody titers in all three measurements taken. The same pattern was also observed for the group of vaccinated elderly, who also showed differences in titer levels over all tests conducted.\n\nThe results of this study are in line with those obtained by Soytas et al.,6 who found that antibody titers were higher in the age group over 80 years, compared to the younger group (≥ 18 and < 60 Years). In the study, efforts were made to compare the level of IgG antibodies in confirmed SARS-CoV-2 patients with RT-PCR. IgG antibody levels and the time required between RT-PCR positive results and antibody levels were recorded. A total of 1,071 patients were divided into two groups, namely group 1 <60 years (n = 902) and group 2 >60 years (n = 169). SARS-CoV-2 IgG antibody titers were higher in group 2 (p = 0.001). These high results appeared in the first three months after RT-PCR detection was positive. Antibody titers were compared by dividing group 2 into three groups by age range (60-69, 70-79 and 80 years); the results showed higher antibody titers in 80-year-old patients (p = 0.044). High levels of COVID-19-specific IgG antibodies may be associated with the severity of the disease.7\n\nThe study conducted by Yang et al.8 evaluated 31,426 blood samples for SARS-CoV-2 antibodies level, showing that IgG levels differed by age group. Data were collected from New York City hospitals from April 9 to August 31, 2020. Semi-stimulative levels of IgG were compared between 85 pediatric patients and 3,648 adult patients. Further analysis of SARS-CoV-2 antibody profiles was performed in serum from 126 patients aged one to 24 years. Of 31,426 antibody test results (19,797 [63.0%] female patients), with 1,119 pediatric patients (average [youngest] age; 11.0 [5.3] years) and 30,232 adult patients (average [SD] age, 49.2 [17.1] years), seroprevalence in pediatric patient populations (197 [16.5%; 95% CI, 14.4%-18.7%]) and adults (5,630 [18.6%; 95% CI, 18.2%-19.1%]) showed similar results. SARS-CoV-2 IgG levels showed a negative correlation with age in the pediatric population (R2= 0.45, p<0.001) and a moderate but positive correlation with age in adults (r = 0.24, p<0.001). Patients aged 19 to 30 years showed the lowest IgG levels (e.g., ages 25-30 years versus 1-10 years: 99 [44-180])).4 Furthermore, studies conducted by Roltgen et al. showed that vaccinated study participants in all age groups (<40 years, 40 to 60 years, >60 years) developed a strong IgG antibody response, although younger individuals yielded higher concentrations of Ig antibodies compared to subjects older than 60.4 years.\n\nRegarding the elderly who had been vaccinated, Muller et al. conducted a cohort study with two age groups, namely with individuals under the age of 60 years and over the age of 80 years, to compare their antibody responses to the first and second doses of BNT162b2 COVID-2019 vaccination by BioNtech (Germany) and Pfizer (USA). Although most participants in both groups produced titers of specific IgG antibodies for the SARS-CoV-2 S protein, the titers were significantly lower in the elderly participants. Although elevated antibody levels after the second immunization were higher in the elderly participants, the average absolute titer level of this group remained lower than that in the <60 age group. After the second vaccination, 31.3% of the elderly (>80 years) had no detectable specific antibodies, compared to the younger group, in whom only 2.2% had no detectable specific antibodies.9\n\nThe effectiveness of vaccines in the elderly has not been widely studied until now. (Weinberger, 2018, Van Jaarsveld, 2020) Markers commonly used to assess immunization effectiveness are antibody titers, antibody isotypes, and the immune system's ability to neutralize pathogens.10 Immunosenescence is a term used to describe decreased immunity with age, encompassing quantitative and qualitative aspects of the immune system response that are likely to impact the safety and effectiveness of the observed vaccine.11 As we grow older, the number of naive T cells available to respond to vaccines decreases (Valiathan et al., 2016). The normal ratio of CD4:CD8 cells increases with age, and becomes much higher in older age, due to a significant decrease in CD8 T cells (McBride and Striker, 2017). Aging also leads to a loss of CD8 and CD4 T cells receptor diversity, and overall reduces T cell viability. Qualitative changes include a shift in lymphocyte production to short-lived effector T cells instead of memory progenitor cells, resulting in a disruption of the response of helper T cells to vaccination.11,12 The number of B cells tends to be constant with age, but reduced expression of certain proteins leads to fewer functional antibodies being produced (Montecino-Rodriguez, 2013). Therefore, theoretically, vaccines may tend to be less effective in the elderly. Furthermore, the relative importance of the cellular aspect of the immune response in COVD-19 is unclear, especially in the elderly, so antibody levels may not be sufficient to induce immunity (Hasan et al., 2021). The impact of immunosenescence on vaccine safety is also uncertain.13 Although the risk of serious side effects mediated by overactivation of the immune system is theoretically lower, the condition is also offset by an increased tendency to general side effects resulting from aging.14\n\nIn our study, there was a difference in antibody titers between the survivor and vaccinated groups in the young age subgroup with no comorbidities, for the first and third tests. The difference was not significant for the second test. In contrast, overall, in the young survivor age group without comorbidities, there was also a difference between titer levels for all three tests conducted. The same was also found in young age group without comorbidities undergoing vaccinations.\n\nThe Ritchie et al.15 study measured antibodies specific to the S protein from patients who had been diagnosed with COVID-19, between January 1 and June 30, 2020, using enzyme-linked immunosorbent assays during the acute and/or convalescent phase. A total of 84 individuals participated in the study. Of those, 19 participants had antibody titers measured during the recovery phase and 65 patients had antibody titers measured during the acute and recovery phases. In 65 patients with two antibody measurements, all had higher antibody titers during the recovery phase. Factors associated with increased antibody titers and subsequent development of antibody titers were evaluated. Significant effects were found for antibody titers and age (p = 0.006), sex (p = 0.06), hypertension (p = 0.07), diabetes mellitus (DM) (p = 0.02), hyperlipidemia (p = 0.009), smoking (p = 0.02), lung disease (p = 0.04), oxygen inhalation during hospitalization (p = 0.02), ventilation management (p = 0.06), use of glucocorticoids (p = 0.03), increase in temperature (p = 0.003), increase in maximum C-reactive protein (CRP) (p <0.001), lactate dehydrogenase (LDH) (p <0.001), and fibrinogen levels (p = 0.01) during the course of the disease. Gradual multivariate analysis showed that male sex (p = 0.04), DM (p = 0.03), and maximum increase in CRP during the acute phase (p <0.001) were associated with increased IgG antibodies. Glucocorticoid use was not associated with antibody titers.15,16\n\nComorbid individuals are a high-risk group for COVID-19, but so far the effectiveness and safety profile of COVID-19 vaccination in that group has not yet been concluded on (Bellido and Peréz, 2021). The study, conducted by Choi et al., compared the effects of comorbid conditions on each vaccine undergoing phases 2 and 3 of clinical trials.15 In phase 2 and 3 clinical trials, the Pfizer COVID-19 vaccine showed that the most commonly found comorbid conditions were hypertension (24.5%), DM (7.8%), and chronic lung disease (7.8%). No significant differences in side effects were found in the comorbid group compared to the total sample population. Six deaths were reported: two in the vaccination group and four in the control group. Of the deaths in the vaccination group, one patient was obese and atherosclerotic, and it was assessed that the deaths were not vaccine-related.17 In phase 2 and 3 clinical trials conducted in the UK and Brazil for the AstraZeneca COVID-19 vaccine (BNT162b2), 39.3% of the sample population had one or more underlying diseases, including obesity with a BMI of 30 kg/m2, cardiovascular disease, lung disease, and DM. Although the preventive effect was slightly lower in patients with one or more underlying diseases (58.3%; 95% CI: 33.6–73.9) compared to the total sample population, the difference was not statistically significant. No deaths were found related to vaccinations.17,18\n\nIn this study, there were no differences in antibody titers between young survivor age groups who had comorbidities compared to those who underwent vaccination. After further analysis, a difference in titers was found between the three tests in the young survivor age group with comorbidities. However, there was no significant difference in antibody titers between the three tests in the young age group with comorbidities who received vaccinations. This difference between the vaccination and survivor groups possibly occurred due to the decrease in the median antibody titers in the survivor group, along with an increase in the median value in the vaccination group.\n\nOverall, in all subjects indiscriminate of their age group, it was seen that there were differences in antibody titers in the survivor and vaccination groups. There was also a decrease in the median value in the survivor group and an increase in the median value of antibody titers in the vaccinated group. During further analysis, titers for both survivor and vaccinated groups also showed differences between the three tests performed. Zhou et al.19 conducted a seroconversion study in 173 patients affected by COVID-19, using tests developed to detect antibodies to the S protein RBD of SARS-CoV-2.11 The median seroconversion time of total antibody seroconversion, IgM, and IgG was 11, 12, and 14 days respectively. Seroconversion rates for total antibodies, IgM, and IgG were 93.1%, 82.7%, and 64.7%, respectively, with cumulative seroconversion curves indicating that total antibody levels and IgM reached 100% 30 days after onset.18,20 Antibody sensitivity goes beyond the RNA test from day 8 after the onset of symptoms and reaches more than 90% on the 12th day after onset.18 Among samples from advanced-phase patients (days 15-39 after onset), sensitivity to total antibodies, IgM, and IgG was 100.0%, 94.3%, and 79.8% respectively. In contrast, RNA was only detected in 45.5% of samples from day 15-39.18 Analysis of 285 patients further supported IgG seroconversion within 19 days after the onset of symptoms.19,21\n\n\nConclusions\n\nBased on demographic data, female patients accounted for the majority at 60.7%. Most of the participants belonged to the non-elderly age group, which was further divided into two more groups, i.e., those living with a comorbidity and those with no comorbidity, with the same number of samples as elderly respondents. The proportion between the group of survivors and vaccinations for both the elderly and nonelderly categories were balanced. Antibody titers in the elderly group of survivors relatively decreased with time. The antibody titer in the elderly group undergoing vaccination relatively increased. Antibody titers in the elderly survivor group and those who underwent vaccination were significantly different for the first test. Antibody titers in non-elderly survivor groups without comorbidities relatively decreased. Antibody titers in nonelderly groups without comorbidities undergoing vaccination were relatively elevated. Antibody titers in non-elderly without comorbidities in both unvaccinated and vaccinated groups were significantly different for the first and third tests. Antibody titers in non-elderly groups with comorbidities in survivors decreased. Antibody titers in nonelderly groups with comorbidities undergoing vaccination relatively decreased. No significant difference in antibody titers was detected between non-elderly survivors with comorbidities and vaccinated non-elderly with comorbidities. In the survivor group, a decrease in antibody levels was found, especially at 56 days and 84 days after COVID-19 diagnosis. In the group undergoing vaccination an increase in antibody levels was shown, especially at 56 and 84 days after the first vaccination. In this study, a significant difference in antibody titers in the first and third tests was found between a group of recovering COVID-19 cases and participants who underwent vaccination. In the entire group, there were significant differences between the three tests conducted, except in nonelderly groups with comorbidities. From this study, it was found that in the survivor group, antibody titers were still found for 84 days after a COVID-19 diagnosis. In the vaccinated group, it was found that antibody titers increased significantly at 56 days after vaccination.\n\n\nData availability\n\nFigshare: Comparison of Immunogenicity Response Between Natural COVID-19 and Post-Vaccination Infections. https://doi.org/10.6084/m9.figshare.16995010.v1 22\n\nThis project contains the following underlying data:\n\n- Raw Data for Comparison of Immunogenicity Response Between Natural COVID-19 and Post-Vaccination Infections.\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\nPal M, Berhanu G, Desalegn C, et al.: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2): An Update. Cureus . 2020; 12(3):e7423. Published 2020 Mar 26. PubMed Abstract \| Publisher Full Text\n\nDinleyici EC, Borrow R, Safadi MAP, et al.: Vaccines and routine immunization strategies during the COVID-19 pandemic. Hum Vaccin Immunother . 2021; 17(2):400–407. PubMed Abstract \| Publisher Full Text\n\nPascual-Iglesias A, Canton J, Ortega-Prieto AM, et al.: An Overview of Vaccines against SARS-CoV-2 in the COVID-19 Pandemic Era. Pathogens . 2021; 10(8):1030. Published 2021 Aug 14. PubMed Abstract \| Publisher Full Text\n\nWu Z, Hu Y, Xu M, et al.:Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine (CoronaVac) in healthy adults aged 60 years and older: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial.Lancet Infect Dis. 2021 Jun;21(6):803–812. Publisher Full Text \| PubMed Abstract \| Free Full Text\n\nMartins Van Jaarsveld G. The Effects of COVID-19 Among the Elderly Population: A Case for Closing the Digital Divide.Front Psychiatry. 2020 Nov 12;11: 577427. Publisher Full Text \| PubMed Abstract \| Free Full Text\n\nSoytas BR, Cengiz M, Islamoglu MS, et al.: Does the COVID-19 seroconversion in older adults resemble the young?.J Med Virol. 2021 Oct; 93(10): 5777–5782. Publisher Full Text PubMed Abstract \|\n\nSchoeman D, Fielding BC: Coronavirus envelope protein: current knowledge.Virol J. 2019 May 27; 16(1): 69, Publisher Full Text PubMed Abstract \|\n\nYang HS, Costa V, Racine-Brzostek SE, et al.: Association of Age With SARS-CoV-2 Antibody Response.JAMA Network Open. 2021 Mar 22; 4(3): e214302, Publisher Full Text PubMed Abstract \|\n\nMüller L, Andrée M, Moskorz W, et al. : Age-dependent Immune Response to the Biontech/Pfizer BNT162b2 Coronavirus Disease 2019 Vaccination.Clin Infect Dis. 2021 Apr 27 [cited 2021 Sep 26]; 73: 2065–2072. Publisher Full Text PubMed Abstract \|\n\nWorld Health Organization: Weeklly epidemiological update on COVID-19. 13 July 2021. Indonesia: World Health Organization; 2021 Jul; p.1–16. Report No.: 48.\n\nWorld Health Organization: COVID-19 Indonesia situation report World Health Organization; 2021 Jul; p. 1–37. Report No.: 63.\n\nPark JW, Lagniton PNP, Liu Y, et al.: mRNA vaccines for COVID-19:what, why and how.Int J Biol Sci. 2021 Apr 10; 17(6): 1446–1460. Publisher Full Text PubMed Abstract \|\n\nSoiza RL, Scicluna C, Thomson EC: Efficacy and safety of COVID-19 vaccines in older people.Age Ageing2020 Dec 50; 50: 279–283. Publisher Full Text\n\nThe pandemic pipeline - PubMed: [cited 2021 Jul 20]. PubMed Abstract\n\nRitchie H, Ortiz-Ospina E, Beltekian D, et al.: Coronavirus Pandemic (COVID-19). Our World in Data. 2020 Mar 5 [cited 2021 Jul 20]. Reference Source\n\nKutsuna S, Asai Y, Matsunaga A, et al.: Factors associated with anti-SARS-CoV-2 IgG antibody production in patients convalescing from COVID-19.J Infect Chemother. 2021 Jun 1; 27(6): 808–813. Publisher Full Text PubMed Abstract \|\n\nHalim M: COVID-19 Vaccination Efficacy and Safety Literature Review.J Clin Med Res. 2021 Feb 2. Publisher Full Text\n\nMahanty S, Prigent A, Garraud O: Immunogenicity of infectious pathogens and vaccine antigens.BMC Immunol. 2015 May 29; 16: 31. Publisher Full Text PubMed Abstract \|\n\nZhou F, Yu T, Du R, et al.: Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study.The Lancet. 2020 Mar; 395(10229): 1054–1062. Publisher Full Text PubMed Abstract \|\n\nJaneway’s Immunobiology - Kenneth Murphy, Casey Weaver - Google Buku. [cited 2021 Jul 20]. Reference Source\n\nRitchie H, Ortiz-Ospina E, Beltekian D, et al.: Coronavirus Pandemic (COVID-19).Our World in Data. 2020 Mar 5 [cited 2021 Jul 20]. Reference Source\n\nKristanto E: Raw Data For Comparison of Immunogenicity Response Between Natural COVID-19 and Post-Vaccination Infections. figshare. Dataset . 2021. Publisher Full Text" }	[ { "id": "124630", "date": "08 Mar 2022", "name": "Anggraini Dwi Sensusiati", "expertise": [ "Reviewer Expertise COVID-19 Research and Radiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 good research and it is needed to make people sure that vaccination is very important. Some comments:\nThe method of this study is clearly reported, but the decision of 30 subjects must be explained.\n\nThe references must be added to make the background more comprehensive.\n\nComorbidity in this research must be explained before the results.\n\n\"No significant difference in antibody titers was detected between non-elderly survivors with comorbidities and vaccinated non-elderly with comorbidities\" this needs more explanation in the discussion based on references.\n\nA short and clear conclusion will be better.\n\nIs the work 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/11-212
https://f1000research.com/articles/10-48/v1	26 Jan 21	{ "type": "Method Article", "title": "Sequencing the pandemic: rapid and high-throughput processing and analysis of COVID-19 clinical samples for 21st century public health", "authors": [ "Megan L. Folkerts", "Darrin Lemmer", "Ashlyn Pfeiffer", "Danielle Vasquez", "Chris French", "Amber Jones", "Marjorie Nguyen", "Brendan Larsen", "W. Tanner Porter", "Krystal Sheridan", "Jolene R. Bowers", "David M. Engelthaler", "Darrin Lemmer", "Ashlyn Pfeiffer", "Danielle Vasquez", "Chris French", "Amber Jones", "Marjorie Nguyen", "Brendan Larsen", "W. Tanner Porter", "Krystal Sheridan", "Jolene R. Bowers", "David M. Engelthaler" ], "abstract": "Genomic epidemiology has proven successful for real-time and retrospective monitoring of small and large-scale outbreaks. Here, we report two genomic sequencing and analysis strategies for rapid-turnaround or high-throughput processing of metagenomic samples. The rapid-turnaround method was designed to provide a quick phylogenetic snapshot of samples at the heart of active outbreaks, and has a total turnaround time of <48 hours from raw sample to analyzed data. The high-throughput method was designed for semi-retrospective data analysis, and is both cost effective and highly scalable. Though these methods were developed and utilized for the SARS-CoV-2 pandemic response in Arizona, U.S, and we envision their use for infectious disease epidemiology in the 21st Century.", "keywords": [ "Genomic epidemiology", "SARS-CoV2", "targeted genomics", "sequencing methods", "phylogenetics" ], "content": "Introduction\n\nWith the advent of rapid and inexpensive next-generation sequencing, genomic epidemiology has proven to be an invaluable resource for the elucidation of disease outbreaks. Extending beyond traditional shoe-leather approaches, rapid-turnaround sequencing methods have allowed researchers to quickly gain insight into the genetic nature of pathogens at the heart of active outbreaks1–6. By monitoring pathogen evolution over the course of an outbreak, large-scale genomics have the potential to allow for transmission mapping for infection control and prevention7,8, to distinguish independent cases from those part of active clusters9, and to identify epidemiological patterns in time and space on both local and global scales7,10–12.\n\nThe most recent example of this has been the collaborative genomic efforts mounted in response to the SARS-CoV-2 outbreak. Not long after the initial cases were identified, whole-genome sequencing quickly established the etiologic agent as a novel coronavirus13, the origins of which have since been elucidated14. Following the rapid spread of SARS-CoV-2, current next-generation technology and analysis pipelines allowed viral sequencing to take place on an unprecedented global scale, with collaborative consortia forming world-wide for the specific purpose of tracking and monitoring the pandemic15–17.\n\nIn most instances, including with the SARS-CoV-2 outbreak, genomic epidemiology has provided a retrospective view of pathogen spread and evolution well after the information is useful in the public health response to the outbreak1,2. Genomic epidemiology should guide contemporaneous outbreak control measures, but can only do so if the data are generated and interpreted in real-time quickly enough to inform a response18. As technology has advanced, the potential exists to move beyond providing a retrospective genomic snapshot of an outbreak months after its occurrence, to providing actionable data in real-time for current outbreaks within hours after cases are identified4. Real-time genomic tracking has already proven valuable in a number of instances, including the recent West-African Ebola outbreak3,18.\n\nThis is not to discredit the value of large-scale, retrospective studies. While rapid-turnaround genomics may prove essential for outbreak containment, retrospective studies will continue to be necessary to track pathogen evolution, gauge success of public health interventions, and to evaluate pathogen/host movement and behavior. With the recent SARS-CoV-2 pandemic, retrospective studies have so-far proven successful in identifying the timing and sources of outbreaks on a local19 and global scale5,20, in evaluating the effectiveness of early interventions5, and in identifying super-spreader events21. Thus, in addition to real-time monitoring, high-throughput, cost-effective sequencing and analysis are needed to gain a better understanding of pandemics.\n\nHere, we report two Illumina-based sequencing and analysis strategies for either real-time monitoring or large-scale, high-throughput targeted genomic sequencing of complex samples. Though these strategies were developed for use with the current SARS-CoV-2 pandemic, we envision their potential use in any situation in which a genomic response is needed.\n\n\nMethods\n\nRemnant nasopharyngeal swab specimens or extracted RNA were obtained from, or received by, the TGen North Clinical Laboratory (TNCL) in Flagstaff, AZ. All samples had previously tested positive for SARS-CoV-2 by RT-PCR.\n\nRNA was extracted using the MagMax Viral Pathogen II kit and a Kingfisher Flex automated liquid handler (ThermoFisher Scientific), with a DNase treatment incorporated to maximize viral RNA recovery from low viral-burden samples, defined as having an RT-PCR cycle-threshold (Ct) value above 33.0. These methods allowed for the rapid, scalable processing of a small number to hundreds of samples at once with minimal personnel, and prevented RNA extraction from becoming a bottleneck to overall throughput (https://doi.org/10.17504/protocols.io.bnkhmct6). Remnant RNA was obtained from TNCL, and had been extracted following their FDA emergency use authorization protocol for the diagnosis of COVID-19.\n\nSARS-CoV2 RNA was amplified for both of the sequencing methods described below following the nCoV-2019 sequencing protocol V.122 and using the ARTIC v3 primer set23. Adapters were added to the resulting amplicons by one of the following means described below. The full sample processing workflow, starting with raw RNA and ending with deliverable data, is illustrated in Figure 1 for reference.\n\nBasic workflow for processing of either high-throughput or rapid-response samples.\n\nFor samples requiring immediate attention, e.g. those from patients potentially involved in an active outbreak, adapters were added with the DNA Prep kit (Illumina) as previously described24. Amplicons were sequenced on the MiSeq platform, using a Nano 500 cycle kit with v2 chemistry (Illumina) (https://doi.org/10.17504/protocols.io.bnnbmdan).\n\nIn instances where retrospective data were needed from large numbers of samples, adapters were added with the plexWell384 kit (Seqwell). Samples were multiplexed in batches of 1,152 and sequenced on a NextSeq 550 with v2 chemistry and 150 X 150 bp reads (Illumina). When batch sizes were not large enough to fill a NextSeq run, samples were sequenced on a MiSeq, with V3 chemistry (Illumina) (https://doi.org/10.17504/protocols.io.bnkimcue).\n\nVirus genome consensus sequences were built using the Amplicon Sequencing Analysis Pipeline (ASAP)25,26. First, reads were adapter-trimmed using bbduk27, and mapped to the Wuhan-Hu-1 genome28 with bwa mem29 using local alignment with soft-clipping. Bam alignment files were then processed to generate the consensus sequence and statistics on the quality of the assembly by the following: 1) Individual basecalls with a quality score below 20 were discarded. 2) Remaining basecalls at each position were tallied. 3) If coverage ≥10X and ≥80% of the read basecalls agreed, a consensus basecall was made. 4) If either of these parameters were not met, an ‘N’ consensus call was made. 5) Deletions within reads, as called during the alignment, were left out of the assembly, while gaps in coverage (usually the result of a missing amplicon) were denoted by lowercase ‘n’s. Only consensus genomes covering at least 90% of the reference genome with an average depth of ≥30X were used in subsequent analyses. Consensus genomes generated using these methods include those from Ladner et al.19. Statistics reported for each sample included: total reads, number of reads aligned to reference, percent of reads aligned to reference, coverage breadth, average depth, and any SNPs and INDELs found in ≥10% of the reads at that position.\n\nRapid phylogenetic inference was used when it was critical to rapidly answer two initial genomic epidemiological questions: i) are the samples in this set closely related, and ii) to what samples in the public database are these most closely related? To answer the former, SNP comparisons were made among a sample set of interest and output in text format directly from the ASAP results, foregoing the computational time of generating phylogenetic trees.\n\nTo answer the latter, a database of all SNPs in the GISAID global collection16 was generated and periodically updated by downloading all the genomes and filtering them for quality and completeness, then aligning to the WuHan-Hu-1 reference to identify any variants, as described30. The list of all variants and metadata for each sample were then put into a relational database for fast querying. The list of SNPs common to samples in a given set was then compared to this global SNP database using a custom script to determine how many GISAID genomes shared all or a subset of the SNPs from the set. A stacked Venn diagram to illustrate globally-shared SNPs was constructed using the statistical package “R” (Version 4.0.2)31 and the “ggplot2”32 and “ggforce”33 R-packages.\n\nFor subsequent, more sophisticated phylogenetic analyses, phylogenetic trees were constructed in NextStrain17, with genomes from GISAID subsampled by uploading our genomes of interest to the UCSC UShER (UShER: Ultrafast Sample placement on Existing tRee) tool34, identifying relevant genomes (those most related to our genomes of interest), and further reducing that set of genomes when necessary with genome-sampler35.\n\nSamples used were remnant, de-identified samples from a clinical diagnostics lab. No ethical approval was required for their inclusion in this study.\n\n\nResults\n\nFor the Seqwell workflow, turnaround time from raw sample to sequence data was approximately 72 hours, and to phylogenetic inference approximately 10 hours, for 1,152 samples (Table 1). Cost per sample was ~$50, which is comparable to similar sample prep methods (Table 1)36. Success rates, defined as the percentage of samples with greater than 90% genome coverage, were similar to other previously described methods36,37. Of a random subset of 897 samples analyzed, approximately 83% of samples with a Ct <33 yielded genomes with ≥90% breadth of coverage of the reference genome, i.e., a complete genome, with an average breadth of coverage of the reference genome of 91%. (Figure 2, Table 2). Failure to obtain a complete genome when Ct was <33 was attributed to sample preparation error, sample degradation, or poor sequencing run metrics. Beyond a Ct of 33, success rate dropped drastically. Between Ct values of 33 and 35, complete genome success rate was ~41% (Table 3), with an average breadth of coverage of 78%. Above a Ct of 35, ~18% of samples yielded a complete genome, and breadth of coverage dropped below 57% (Table 2).\n\nWorkflow metrics for Illumina’s DNA prep method, and Seqwell’s plexWell method.\n\n* Listed is the number of samples that can be processed within the specified turnaround time, on a single sequencing run. This is not necessarily the upper limit of either processing system.\n\n** Time from raw sample to analyzed data\n\nA. Nucleocapsid-2 Ct value vs percent genome coverage and B. percent total reads mapped to SARS-CoV-2 for 897 SARS-CoV-2-positive samples sequenced using Seqwell’s plexWell 384 system.\n\nAverage sequencing metrics for various RT-PCR cycle threshold values of samples sequenced using the plexWell 384 system, and either an Illumina MiSeq or an Illumina NextSeq550.\n\nCt value reported is that for the nucleocapsid-2 gene\n\nUniform depth of coverage was targeted for all samples\n\nAverage sequencing metrics for various RT-PCR cycle threshold values of samples sequenced using the Illumina DNA Prep system and an Illumina MiSeq.\n\nCt value reported is that for the nucleocapsid-2 gene\n\n**Uniform depth of coverage was targeted for all samples\n\nUniform depth of genome coverage across samples was targeted when pooling samples for sequencing, but depth generally decreased as Ct increased. Average depth of coverage was approximately 2766X through a Ct of 33. Past 33, coverage dropped off sharply, with an average depth of 1221X between Ct values of 33-35. Between Cts of 35 and 37, coverage was 716X, and above a Ct of 37, coverage dropped to 576X (Table 2).\n\nSimilarly, percent of total reads aligning to the SARS-CoV-2 reference genome decreased as Ct increased (Table 2, Figure 2). Up to a Ct of 30, most (~96%) of reads mapped to SARS-CoV-2. From Cts of 30 to 35, this noticeably decreased, and continued to drop as Cts rose. Beyond a Ct of 37, average percentage of reads aligning dropped to 38%.\n\nTurnaround time for the Illumina DNA prep method was significantly faster than the high-throughput plexWell system. It took less than 48 hours to go from raw sample to deliverable, analyzed data (Table 1), and this time could potentially be reduced further by reducing cycle numbers per sequencing run.\n\nBoth the plexWell and DNA Prep methods use a tagmentation system for adapter addition, rather than a ligation-based approach. This results in adapters being added ~50 bp or more from the end of overlapping amplicons generated during gene-specific PCR. A second PCR step amplifies only the tagmented regions, resulting in final libraries of ~300bp. These approaches negate the need for primer trimming prior to alignment.\n\nGenerating consensus genomes and a SNP report from the sequence data, which takes approximately 15 minutes for a small (<=24 samples) dataset, quickly shows whether the samples are part of the same outbreak or transmission network. To quickly find a potential origin of a cluster or sample (assuming genomes in the public domain were collected prior to the sample(s) of interest), a global SNP database can be used. Constructing or updating a global genome SNP database takes several hours, but it can be done prior to sample sequencing (and regularly). Running the script to query the global SNP database for particular SNPs of interest takes mere seconds.\n\nTo rapidly visualize results of the global SNP database query, a stacked Venn diagram (aka, “onion diagram”) visually describes the hierarchical nature, i.e., the parsimony, of SNPs found in the SARS-CoV-2 genomes (Figure 3), and is easily generated using the methods described above or an alternative tool.\n\nStacked Venn “onion” diagram indicating the hierarchical nature of cluster-specific SNPs relative to the reference strain in global collection of SARS-CoV2 samples.\n\nAlthough fewer data are available from the Illumina DNA prep method, as it was primarily used to process 5 or fewer samples at a time, data suggest that it performed slightly worse than the Seqwell system, with odds of obtaining a complete genome dropping to 0 at Ct values greater than 30 (Table 3).\n\n\nDiscussion\n\nThe recent SARS-CoV-2 outbreak has highlighted the need for real-time sequencing and data analysis capacity in the face of active pandemics, as well as high-throughput sequencing and analysis strategies for comprehensive retrospective analysis and evaluation. We describe two different strategies that can be used in combination with Illumina platforms for the rapid or high-throughput interrogation of samples involved in disease outbreaks. Though the results reported here are specific to the SARS-CoV-2 outbreak, these methods could conceivably be modified and applied to many other situations in which a genomic epidemiological response is needed.\n\nThe Illumina DNA prep-SNP-comparison analysis method is effective in providing rapid sequence data and genomic epidemiology information for small numbers of samples (<48 hours from raw sample to rough phylogenetic placement). Though throughput is limited by both the number of available indices for multiplexing and the nature of the protocol itself, this method has the advantage of being scalable to small numbers of samples, and can be performed in the course of several hours for small sample subsets.\n\nThe Seqwell plexWell system provides a scalable, cost-effective, and high-throughput method of processing thousands of samples with minimal laboratory personnel (Table 1). As the plexWell protocol calls for the pooling of samples at an initial step in the adapter tagmentation process, hundreds of samples were able to be taken through the later steps of the protocol by a single individual in an 8-hour timeframe. This, coupled with the availability of thousands of index combinations, allowed for a high-throughput, cost-effective means of processing large numbers of samples on a weekly basis with minimal laboratory personnel and infrastructure.\n\nTypical analyses of virus genomes include phylogenetic tree construction to understand transmission patterns. Nextstrain17 and GISAID16 have been crucial to the SARS-CoV-2 scientific community for global and local epidemiologic understanding, and tools for smart subsampling (e.g. genome-sampler35) are now necessary with the growth of the public databases. However, reconstructing phylogenies, especially paired with finding relevant subsets, takes time, and is often overkill for initial, time-sensitive public health needs. We employ a simple, rapid analysis method and visualization meant as a quick-look to determine relatedness among a sample set of interest and/or relatedness of a sample or set to the entire public database of genomes. Because of the novelty of SARS-CoV-2 and its low rate of recombination, merely comparing the low number of SNPs across samples without applying a phylogenetic model or program is often enough to answer initial questions about COVID-19 transmission, e.g. whether samples in a given set are closely related, and/or which samples in the global database are most closely related to a given sample set. Our SNP queries followed by generation of a stacked Venn diagram (onion diagram) offer a much faster alternative or antecedent to complete phylogenetic analysis. Other pathogens or situations where SNP numbers are expected to be very low may also benefit from these rapid analysis methods.\n\nFor retrospective studies, time can allow for more robust phylogenetic analyses including smart subsamplers35, such as NextStrain17 and other commonly used phylogenetic tools; however their employment can significantly add time to a rapid response. The UShER tool34, which can rapidly place genomes onto an existing SARS-CoV-2 phylogenetic tree, can greatly speed-up the final analysis. Parsing the output of UShER generates a subset of public genomes that are phylogenetically close to the samples of interest. This reduces the input dataset to subsamplers such as genome-sampler35, which significantly reduces the computation time for further subsampling based on geography and time, which in turn significantly reduces the computation time for a NextStrain analysis.\n\nEach of the two methods have their limitations. We observed a reduced success rate of the rapid Illumina DNA Prep method over the high-throughput Seqwell system, as evidenced by both decreased overall breadth of coverage and decreased success of obtaining complete genomes at Ct values above 30. It should be noted, however, that rapid prep study was conducted on a limited sample set, using samples from active outbreak clusters that were shipped from long distances through varying ambient temperatures. Samples used to evaluate the Seqwell system were obtained locally and processed entirely in-house. This difference in handling, coupled with the sample size difference, may in part account for the differences in results in the two prep methods. Also, at ~$100/sample in reagent costs, the rapid Illumina DNA Prep method is less cost-effective (Table 1). And though the turnaround for the protocol is ~1.5 days, faster sequencing is achievable through other methods, such as through the use of long-read Nanopore sequencing18,36. Nanopore technology, however, has the disadvantage of having a higher per-base error rate when compared to short-read sequencing methods18, thus its lack of accuracy may outweigh any potential time savings, particularly in situations where relatively few nucleotide variants can radically alter phylogenetic placement such as for SARS-CoV-2. Also, the SNP-comparison analysis is rapid and robust because of the relatively low numbers of SNPs so far documented in the SARS-CoV-2 genome, due to the virus’s novelty, the lack of recombination, and the unmatched robustness of the global SARS-CoV-2 genome database. Though this method could be applied to other types of outbreaks, rapid, precise phylogenetic placement will rely on these same factors.\n\nThe high-throughput SeqWell prep system also has its drawbacks. The pooling strategy of the plexWell system prevents downscaling, thus small numbers of samples cannot be processed effectively with this system. The turnaround time of this method, when large sample numbers are processed, is not competitive36. The majority of processing time is lost to the ARTIC portion of the protocol, however, and not specifically to the plexWell adapter addition. And though the combinatorial index system allows for thousands of samples to be multiplexed in a single sequencing run, the SARS pandemic has demonstrated that even this may not be sufficient to meet the data challenges presented by expansive disease outbreaks.\n\nDespite overwhelming benefits of employing next generation technological advances in real-time during a public health emergency, challenges remain when using genomic epidemiology as a means of pandemic control and monitoring. It has been demonstrated that genomics are not, in and of themselves, sufficient to completely elucidate the mechanisms and transmission of all pathogen-transmitted disease, particularly when asymptomatic and mild infections are known to play a role in transmission38 but are less likely to be identified and subsequently sequenced. Thus, the integration of genomic and traditional epidemiology is paramount to the success of this 21st century public health capability.\n\n\nData availability\n\nZenodo: Raw sequencing metrics from two different prep methods for obtaining SARS-CoV2 genomes, http://doi.org/10.5281/zenodo.430990039.\n\nThis project contains the following underlying data:\n\nSequencing_metrics_IlluminaDNAprep.xlsx- data used for generation of Table 3\n\nSequencing_metrics_Seqwellprep.xlsx- data used for generation of Figure 2 and Table 2\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank Hayley Yaglom for her helpful review of the first draft and the members of the Arizona COVID Genomics Union (ACGU), for their insight and support of this work.\n\n\nReferences\n\nGardy JL, Naus M, Amlani A, et al.: Whole-Genome Sequencing of Measles Virus Genotypes H1 and D8 During Outbreaks of Infection Following the 2010 Olympic Winter Games Reveals Viral Transmission Routes. 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Publisher Full Text\n\nBolyen E, Dillon M, Bokulich N, et al.: Reproducibly sampling SARS-CoV-2 genomes across time, geography, and viral diversity [version 2; peer review: 1 approved, 1 approved with reservations]. F1000Res. 2020; 9: 657. Publisher Full Text\n\nPaden CR, Tao Y, Queen K, et al.: Rapid, Sensitive, Full-Genome Sequencing of Severe Acute Respiratory Syndrome Coronavirus 2. Emerg Infect Dis. 2020; 26(10): 2401–2405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaurano MT, Ramaswami S, Zappile P, et al.: Sequencing identifies multiple, early introductions of SARS-CoV2 to New York City Region. medRxiv. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeredith LW, Hamilton WL, Warne B, et al.: Rapid implementation of SARS-CoV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study. Lancet Infect Dis. 2020; 20(11): 1263–1272. PubMed Abstract \| Publisher Full Text\n\nFolkerts ML: Raw sequencing metrics from two different prep methods for obtaining SARS-CoV2 genomes [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4309900" }	[ { "id": "78623", "date": "16 Mar 2021", "name": "Bronwyn L. MacInnis", "expertise": [ "Reviewer Expertise Virology", "Genomics", "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 a methodologically sound study describing complementary approaches for generating and analyzing SARS-CoV-2 genomic data. The workflows address two common use cases: rapid turnaround of a relatively small number of samples, i.e. for cluster investigation, and larger scale but less urgently time sensitive sequencing.\n\nThe approach described here is sound and there is sufficient detail to follow the methodology.\n\nMy main issue with the manuscript is that it is not clear what the novel contribution is, since each of the approaches are previously described, using commercial and published methods. However it is nonetheless helpful to have the workflows mapped out as they are here, and to benchmark the differences between the approaches. This could be particularly helpful to the many labs now attempting viral/SARS-CoV-2 sequencing for the first time.\n\nI would also advise that the focus on retrospective sequencing limits the potential value of the larger scale approach. It could equally be relevant for larger scale surveillance sequencing, i.e. for variants of concern, real time (or near real time).\n\nI would like to see the costs described in more detail, and in relative terms, since costs of consumables and effort can vary widely across contexts. Please clarify what is included in costs in more detail, whether costs include effort, what the impact of effort is.\nPersonally I find the title to be overstated for the content of the manuscript. I would suggest a title that more clearly conveys that this is a methods paper. In particular I would delete the prefix \"Sequencing the pandemic\", as this suggests something being done at a global scale, and the \"21st century public health\" as there is no direct application to public health.\n\nFinally, although this study clearly focuses on lab methods for sequencing, given the framing I would like to see it acknowledge that currently many of the delays in SARS-CoV-2 sequencing are not in the sample preparation and sequencing steps, rather in processes up and downstream of sequencing.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "7702", "date": "21 Feb 2022", "name": "Megan Folkerts", "role": "Author Response", "response": "Dr. MacInnis, We want to thank you for taking the time to review our paper and provide valuable insight and comments aimed at strengthening our manuscript. We have carefully considered the comments, and have done our best to address each of them. We hope that the revised version of this manuscript addresses the concerns you voiced, and now meets your standards. The authors welcome further constructive criticism. Attached is a point-by-point response to both reviews. Changes made to the manuscript are clearly stated, with line numbers included for your quick reference. Sincerely, Megan Folkerts, MS mfolkerts@tgen.org Translational Genomics Research Institute, North Response to Reviewer 1 (Dr. Bronwyn MacInnis) Comment 1: It is not clear what the novel contribution is Response: We appreciate this valuable feedback and hope we have provided some clarity. This manuscript seeks to provide guidance to labs seeking to undertake rapid and or high throughput sequencing, and it reports the first use of the SeqWell plexWell system for use in conjunction with the commonly used ARTIC primer system. This novel method required few laboratory personnel and minimal equipment, and allowed for the rapid sequencing of thousands of samples per week, a novel pace for such a complex procedure. The manuscript has been reworded to highlight these developments. Changes mentioned here are reflected in the following lines of the manuscript: Abstract: The high-throughput method, first reported here for SARS-CoV2, Lines 45-46: The plexWell method is a novel means of sequencing adapter addition to facilitate high-throughput sample processing. Lines 315-316: Here, we report the first use of plexWell for post-amplification adapter addition for whole genome sequencing of SARS-CoV2. Lines: 321-325: With just two full-time staff members devoted to sequencing, and 2-4 part-time staff responsible for arraying samples prior to the ARTIC/plexWell protocol, 1152 samples were able to be successfully processed and sequenced each week. Thus, the number of genomes completed per full-time (40 hour) employee per week is 384, a novel pace for what is normally a lengthy protocol Comment 2: Focus on retrospective sequencing limits the potential value of the larger scale approach. Response: We agree with this comment; however, sequencing was not merely done retrospectively. All data shown for the rapid-turnaround method were done in real-time, and data were delivered fast enough to inform a public health response. The manuscript has been edited to make this clear via the following (which includes a citation of the relevant MMWR report for this study). Lines 352-355: The data presented here using the rapid-turnaround method were obtained from patients who were part of an active SARS-CoV2 outbreak, and the resulting phylogenetic placements were successfully used to inform public health efforts to determine the potential origin of the outbreak, and to prevent further spread45. Comment 3: I would like to see the costs described in more detail. Response: We have now provided a breakdown of sequencing costs in the supplementary methods. This includes the manufacturer’s reagent pricing, and represents the relative cost to the user minus any institution-specific discounts or advantages. Comment 4: I find the title to be overstated Response: We now recognize that the title overstated the scope of this methods paper. We have changed the title to more closely represent the content of the publication. Comment 5: I would like to see it acknowledged that currently many of the delays in SARS-CoV2 sequencing are not in the sample preparation and sequencing steps, rather in the processes up and downstream of sequencing. Response: We agree that delays aren’t just in sequencing, and have acknowledged and described the other areas of potential delay in the manuscript, as per: Lines 398-407: This paper highlights the potential for rapid turnout of genomic data from epidemiological samples, giving genomics the potential to become invaluable in pandemic response. It is worth noting, however, that sequencing isn’t the only potential bottleneck in the process. For data to be delivered in real-time, coordination between testing sites, sample delivery, and sequencing laboratories must be robust, so that preparation for sequencing can begin shortly after a sample has tested positive for the pathogen of interest. Failures anywhere in this chain can lead to significant delays, which negates the utility of a real-time sequencing pipeline for investigation. Thus, in addition to improvements in the described sequencing and analysis pipeline, coordinating the efforts of testing sites, public health partners, and sequencing laboratories is critical for this technology to be most effective." } ] }, { "id": "92632", "date": "21 Sep 2021", "name": "Keith Crandall", "expertise": [ "Reviewer Expertise phylogenetics", "bioinformatics", "computational biology", "infectious disease" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study proposes a reasonable approach to rapid assessment of viral diversity for SARS-CoV-2 taking advantage of the latest in Illumina sequencing kits and associated prep protocols as well as open source data and tools for downstream analyses. There are no statistics involved and therefore users have no idea how reliable any result might be coming out of such an approach. I would personally prefer public health decisions be based on accurate as well as rapid information. This paper speaks to rapid, but does not speak to accurate. Additionally, the paper describes some methodology that is unique to this work, but the authors do not provide any access to these crucial components. Therefore, as presented, the work seems to not be repeatable. This would need to be fixed before indexing. I have detailed these issues below.\nOne of the biggest issues we have when consulting with individuals or groups interested in SARS-CoV-2 sequencing is deciding on read quantity. The methods discusses 'coverage' for confidence in genome assembly, but there is no discussion at all on the number of reads targeted for sequencing. What is the target number of reads for the SNP analysis versus the whole genome analysis? Can you provide power calculations for identifying SNPs based on read coverage? Quality of assembled genomes based on read coverage? I think such analyses would be particularly helpful to individuals trying to implement this strategy in their own labs.\nIs there a link to the 'global SNP database'? Is this an open access resource? If so, you should provide a link to it. If not, then the paper is not particularly useful because the work is not replicable.\nIs the 'custom script' for SNP identification available? This should also be available via something like GitHub for reproducibility.\nIt's too bad the authors didn't go through the effort of consenting the sample donors and including clinical data. The genomic information is highly useful (as outlined in the paper), but the genomic information COUPLED with the EHR and epidemiological data would be infinitely more useful.\nThere don't seem to be any statistics associated with 'transmission network' assignment. How accurate is this inference and how is a user supposed to know about the accuracy of such an assignment?\nSimilarly, there are no statistics associated with the phylogenetic placement of genomes. What is the confidence a user would have in such placement?\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "7703", "date": "21 Feb 2022", "name": "Megan Folkerts", "role": "Author Response", "response": "Dr. Crandall, We want to thank you for taking the time to review our paper and provide valuable insight and comments aimed at strengthening our manuscript. We have carefully considered the comments, and have done our best to address each of them. We hope that the revised version of this manuscript addresses the concerns you voiced, and now meets your standards. The authors welcome further constructive criticism. Attached is a point-by-point response to both reviews. Changes made to the manuscript are clearly stated, with line numbers included for your quick reference. Sincerely, Megan Folkerts, MS mfolkerts@tgen.org Translational Genomics Research Institute, North Response to Reviewer 2 (Dr. Keith Crandall) Comment 1: There are no statistics involved. Response: We agree that this was an oversight, even for a methods paper. All sequencing metrics are now reported in terms of mean, standard deviation, and 95% confidence interval. Comment 2: There is no discussion at all on the number of reads targeted for sequencing. Response: Thank you for this feedback. We have added read counts to all sequencing data metrics within the manuscript body. This is reported in the following paragraphs: Lines 217-229: The number of reads targeted per sample for the high-throughput method was approximately 348,000, considering the limitation of the number of index combinations (i.e., samples per run) that were available at the time. In practice, this varied considerably. The average read count for samples using the plexWell method was 411,375, with 393,925[MOU2] being the average number of reads that aligned to the WuHan-Hu-1 reference strain. Below a Ct of 33, average aligned reads was 475,310 (SD 260292, 95%CI 455789-494831) per sample. Between Cts of 33 and 35, the average reads aligned was 204,234 (SD 175947, 95%CI 163308-245160) per sample. Above 35, read counts dropped off sharply, with a mean of 99,388 reads aligning (SD 188499, 95%CI 68493-130283) per sample. Note that all samples were put through an equimolar pooling step by the plexWell system. This suggests this pooling is not entirely effective when Cts vary considerably in a given sample set. This was confirmed through Kruskal Wallis on the total (unaligned) read counts in each of the above-mentioned groups (p <0.001) Lines 289-296: A minimum of 42,000 reads were targeted for the Illumina DNA prep method, but as sample sizes were small for the outbreak in question, the average aligned reads per sample was much higher. For samples with Cts below 25, the average number of reads aligned to the reference was 275,258.7 (SD 136,816.2, 95%CI 165,784.9-384732.4). Between Cts of 25 and 30, average reads aligned was 200,810 (SD 86,999.43, 95%CI 140,523.6-261096.4). Below a CT of 30, average aligned reads dropped to 116,888 (SD 275257.7, 95%CI 53,002.2-180,773.8). Kruskal-Wallis revealed no significant difference between average aligned reads between these groups (P= 0.08). Comment 3: What is the target number of reads for the SNP analysis vs the WGS analysis? Response: The SNP analysis was run concurrently with the WGS analysis, and the read depth targeted for the SNP analysis is the same as that of the WGS analysis. Targeted read depth, and achieved read depth, has been added to the manuscript (see response to comment 2). A previously published study reported a depth of 10 reads sufficient for SNP calling of SARS-CoV2 genomes (Ladner et al 2020). In all instances, SNPs called using the methods published here far exceed this threshold. Average read depth per sample can be found in the supplementary data. Comment 4: Can you provide power calculations for identifying SNPs based on read coverage? Response: We agree that providing power calculations for SNPs based on read coverage is critical for phylogenetic analyses. The purpose of this paper is simply to report sequencing and analysis methods for the purpose of generating data that can then be fed into more thorough phylogenetic analyses, as we have done with Ladner et al 2020. This is reflected in the following: Lines 122-123: Consensus genomes generated using these methods include those from Ladner et al. 19 . and are similar to those in Peng et al.43 and Stoddard et al.44 Comment 5: What is the quality of assembled genomes based on read coverage? Response: Average depth of coverage, as it correlates to CT value and percent genome coverage, is summarized in Table 2 and 3. Average depth by sample across the same metrics is available in the Supplementary Methods. Comment 6: Is there a link to the global SNP database? Response: The global SNP database, which contains data directly downloaded from GISAID, is not able to be shared via secondary publications, as per GISAID’s data sharing agreement. The contents of the database are described, with one table generated using the Chan et al.35 analysis on genomes downloaded from GISAID, and the other table generated directly from GISAID data which is available for public download from GISAID’s website, which is linked in the reference section. This is addressed via the following: Lines 138-143: The format of this database was simply one table of SNPs, as generated by Chan et al.35, with columns for GISAID ID, position, reference base, and variant base, and a second table of metadata which contained the exact data fields, as downloaded from GISAID. The two tables were linked by the GISAID ID number. The GISAID database cannot be shared via secondary publications as per the database access agreement, however the database is available for download by the public from GISAID’s website16. Comment 7: Is the custom script for SNP identification available? Response: The custom script consisted of a simple set of commands, described in detail in the following so that they can be reproduced: Lines 143-147: The list of SNPs common to samples in a given set was then compared to this global SNP database by first querying the number of global genomes containing each of the individual SNPs, sorting them from most common to least common, then further querying for number of global genomes containing combinations of SNPs by adding in each successive SNP in turn. Comment 8: Genomic information coupled with the epidemiological data would be infinitely more useful Response: We completely support this comment. However, this was not done here as it exceeded the purpose of this paper, which was to provide methods for rapid and high-throughput sample processing and initial phylogenetic placement of genomes. We agree that obtaining patient metadata can provide for a more robust analysis and meaningful interpretation for a more complete epidemiological picture, and in our publications where that is the goal, we make every effort to arrange this. Comment 9: There don’t seem to be any statistics associated with the “transmission network” Response: Thank you for this feedback. This language was changed to reflect the scope of the paper. We were not conducting full transmission analyses (as we overstated in the first draft), but rather, were using the obtained genomic data to guide field epidemiology. Current terminology reflects this, as per: Lines 260-262: Generating consensus genomes and a SNP report from the sequence data, which takes approximately 15 minutes for a small (<=24 samples) dataset, quickly shows whether the samples are part of the same outbreak. Comment 10: There are no statistics associated with the phylogenetic placement of genomes. Response: This is correct. We have cited our previous phylogenetic analyses and other published works that have used and described similar methods. The purpose of this manuscript is primarily to report methods of generating data that can be fed into more complex phylogenetic analyses. We agree that statistics associated with placement are critical, and we have detailed complete phylogenetic analysis of SARS-CoV-2 genomes in Ladner et al. 2020, which follows the published methods of Bolyen et al 2020. Detailed phylogenetic analyses methods and results are not included here, as we felt that this was not part of this study. We have indicated this in the following: Lines 336-340: However, reconstructing phylogenies, especially paired with finding relevant subsets, takes time, and is often overkill for initial, time-sensitive public health needs. We employ a simple, rapid analysis method and visualization meant as a quick-look to determine relatedness among a sample set of interest and/or relatedness of a sample or set to the entire public database of genomes. Lines 349-351: This is not meant to replace more thorough phylogenetic analysis, but rather, to provide a quick genomic snapshot that can inform a public health ground response" } ] } ]	1	https://f1000research.com/articles/10-48
https://f1000research.com/articles/9-159/v1	03 Mar 20	{ "type": "Research Article", "title": "Characteristics of back pain in young adults and their relationship with dehydration: a cross sectional study", "authors": [ "Faizan- ul-Haq", "Uzair Yaqoob", "Muniba Mehmood", "Adeel Ahmed Siddiqui", "Syed Muhammad Usama", "Syed Zohaib Maroof Hussain", "Muhammad Mannan Ali Khan", "Faizan- ul-Haq", "Uzair Yaqoob", "Adeel Ahmed Siddiqui", "Syed Muhammad Usama", "Syed Zohaib Maroof Hussain", "Muhammad Mannan Ali Khan" ], "abstract": "Background: Low back pain (LBP) is one of the major factors impairing the quality of life and is the most frequent cause of disability. Inadequate water intake is believed to be the predisposing factor for LBP particularly in the younger population. It is commonly seen that the incidence of LBP has been on the rise in people between 20-40 years of age. Thus, the basic aim of this study is to find a potential relationship between dehydration and LBP among young adults. Methods: This cross-sectional study was conducted from the medical students and practicing doctors of 21-39 years from March-May 2019. Characteristics of pain along with the daily activities of patients were assessed. The severity was assessed by using the Graded Chronic pain scale (GCPS). Results: Out of a total of 426 participants, 84.74% had LBP. Of these, 44.3% complained of having it more than once a week, with duration usually between 1-7 days. More than half of the patients had their routines disturbed because of this pain. Most of the participants complained of an episodic increase in summers. The majority (75.9%, n=274) drank 5-9 glasses of water a day, 64.5% of them were of opinion that their daily water consumption was enough, while 61.5% felt an association between dehydration and LBP. According to the GCPS, one-third of the population had chronic pain of grade I and the other third had grade IV. Conclusion: It can be concluded that with the increase in the pace of life many individuals who belong to the above mentioned age group have a reduced intake of water, and due to a probable relationship between LBP and dehydration, this might be a reason of the increasing propensity of LBP in them. There is a need for further work in this regard.", "keywords": [ "Back pain", "Dehydration", "Orthopedics", "young adults" ], "content": "Introduction\n\nLow back pain (LBP) is a severely debilitating condition which not only impairs quality of life but also affects the productivity of an individual. In a study published by the World Health Organization (WHO), LBP was recognized as the leading cause of disability1. Out of several causative reasons for LBP, some predominant ones are bad posture, obesity, and low physical activity2. Back pain is emerging as a common complaint among the young population. A study conducted in Finland suggested it as a frequent problem in adults who are in their 30s, increasing in severity with age, evolving as a non-specific and radiating LBP3, which results in reduced productivity and absence from the workplace4. This is the reason why back pain has now emerged as a socioeconomic burden for both the individual and society as a whole5.\n\nIt is believed that inadequate water intake is a predisposing factor for many acute medical conditions and a potential association with some chronic conditions exists. Even a short duration of fluid restriction can lead to loss of body mass, reduced levels of alertness and concentration, tiredness, headaches, and back pain6,7. Secondly, a potential relationship is believed to exist between dehydration and LBP, and this hypothesis is somewhat supported by a study conducted on the hydration of nucleus pulposus and its relation to intervertebral disc derangement. It states that the proper functioning of an intervertebral disc depends upon the nucleus pulposus. Around 80–88% of the nucleus pulposus is composed of water in early life, which gradually decreases with age. The study also reveals that dehydration and degeneration of nucleus pulposus are linked8. Suitable intake of water and maintaining adequate fluid intake is necessary as dehydration can affect the body’s working capacity in many ways, one of which is by the reduction of optimum blood flow to the body’s core muscle7. Also relevant is the role of adequate hydration on postural control, which has a pivotal role in our daily life, especially in our biomechanical wellness, which can become a cause of musculoskeletal problems including LBP if not functioning properly. A study published in the International Journal of Neuroscience suggested that adequate fluid intake may help in maintaining posture by preserving the muscles responsible for postural control9.\n\nWater is a vital component for life, constituting 75% of the body weight of infants and 55% of the elderly10. Body fluid and electrolyte homeostasis is dependent on the balance between water intake and output, and around 5–10% is renewed daily11. Moreover, it is essential to maintain an adequate fluid balance, in order to carry out our vital bodily functions12. Regulation of water balance is mainly by passing it through urine, as described by Engell D et al.13. Daily fluid consumption of a human body mostly exceeds its daily requirement and this may change in accordance with the level of physical exertion, climate, and several other factors that affect the water balance14. The pace of modern life has generally led to it becoming more difficult to fulfill body water demands, which leads to dehydration15. Clinically the term dehydration denotes body water loss, either without salt or along with it15. It is observed that reduced thirst, reduced water intake, and thermal dehydration are the major predisposing factors for dehydration and the situation becomes worse when these factors are combined with reduced renal water-conservation capacity, especially in older subjects16. This point was further emphasized in a study by Armstrong et al., where it was concluded that around 1–2% of pre-exercise body mass is lost, even after a small amount of body water loss17.\n\nIndividuals of 20–40 years of age can be rightly termed as the workforce of our society. They are active in sports, aerobics, workouts, as well as other forms of physical activities, which makes them vulnerable to dehydration. Thus, the basic point on which this study is hinged upon is to establish if there is a potential relationship between dehydration and LBP among the individuals aged 20–40 in the general population; evidence of which may aid in alleviating the socioeconomic burden caused by LBP and improve quality of life.\n\n\nMethods\n\nThis cross-sectional study was conducted at a medical college and a tertiary care hospital from from March to May 2019. Proformas were distributed during lectures and workshops. The sample size was calculated using OpenEpi software version 3.01. Considering an estimated prevalence of LBP in young adults as 42.4%, confidence level of 95%, and a design effect of 1, the minimum sample size was kept as 376 and in the assigned time period of three months, a total sample of 426 was collected18.\n\nInclusion criteria was male and female medical students and young practicing doctors of 21–39 years of age. Those with a Vitamin D deficiency or a joint disease (if disclosed in history) were excluded. For vitamin D deficiency, the participants were sent to the institutional laboratory where blood samples (3–5 mL) were collected by an experienced phlebotomist in gel tubes. The serum was centrifuged at 3000 bpm for five minutes and stored at -80°C for subsequent analysis. Serum vitamin D levels were measured by commercially available enzyme linked immunosorbent assay (ELISA) kits (kit cat#KAP197 by DIA source immunoassays S.A. Belgium). Only ten patients had vitamin D deficiency (considered deficient when it was <12 ng/mL) out of 43619, which lead to the final sample size of 426. Data was collected through a pre-designed proformas in English made by reviewing similar studies from the literature and a short pilot study conducted over two weeks, with 50 participants20–25. Variables included sociodemographics (age, gender, height, and weight), those related to pain (frequency, duration in the last six months, effect on sleep and routine activities, measures to relieve pain and visits to a healthcare professional, and seasonal changes in pain), daily activities (driving, riding, consumption after strenuous activity, duration load of routine work, regular outdoor sports, and weight lifting or aerobic activities, and daily glasses of water drank). Patients’ opinion about their daily consumption of water and the relation of back pain with dehydration was also enquired. The severity of pain was assessed by using the Graded Chronic pain scale (GCPS). It is a seven-item instrument designed to evaluate the severity of chronic pain based on its intensity and disability, with three subscales i.e. characteristic pain intensity score (calculated by combining the current pain intensities, with the worst and average pain status in the last six months), disability score (mean score of the interference of daily living by the pain), and disability points (calculated by combining the number of disability days and the disability score). Based on these scores a combined score of pain and disability is computed, categorizing patients into five categories as Grade 0, no pain; grade I, low intensity and low disability; grade II, low disability and high intensity; grade III, high disability and moderate limiting intensity; and grade V, high disability with severely limiting intensity22. Participants filled the proformas themselves during their lectures and workshops (with proper guidance of investigators) and weight and height were measured by the principal investigator using a scale and measuring tape. They were informed that all information was to be kept confidential. Questionnaires were given an identification number. The data collection procedure was supervised by the principal investigator. Data were entered and analyzed using SPSS v. 22 (IBM Corp., Armonk, NY, US). Frequency and percentages were calculated for all variables, while the mean and standard deviation for continuous variables.\n\n\nResults\n\nOut of a total of 426 participants, 361 (84.74%) had back pain at least once in the last six months. The proportion of males and females was almost equal and the majority (41.3%, n=176) were in the age group of 21–30 years as shown in Table 1 (See underlying data26). Height and weight distribution can also be seen in the table.\n\nMost of the individuals (44.3%, n=160) complained of having back pain more than once a week, with a duration usually (in 68.1%) between 1–7 days. Of these, 59.0% (n=213) of the participants had their sleep affected by the pain, and 59.6% (n=215) were unable to do their routine activities because of this pain. Early measures to reduce pain included medicine (61.77%, n=223) and rest (43.76%, n=158), and more than half of those having back pain used pain killers regularly. The frequency of participants visiting a healthcare professional to consult about this pain is fairly equal to those not doing so. Some seasonal variation in the pain was reported, with most complaining of an episodic increase in summers (36.8%, n=133), while many reported of the pain remaining the same in intensity throughout (36.0%, n=130) (Table 2; see underlying data26).\n\nThe authors also asked about the daily activities of the participants: 57.1% (n=206) reported daily driving; 10.8% (n=39) daily riding; 13.3% (n=48) playing outdoor sports regularly; 4.4% (n=16) regular weight lifting; and 21.6% (n=78) performing regular aerobic exercises. Further data on participant activities are presented in Table 3 (See underlying data26). When asked about their daily water consumption, most (75.9%, n=274) drank 5–9 glasses (of 250 ml) of water a day.\n\nThe majority (64.5%) of the participants were of the opinion that their daily water consumption was adequate, and most (61.5%) felt an association between dehydration and back pain (Table 4).\n\nWe applied the graded chronic pain scale to assess the severity of back pain where around one-third of the population had chronic pain of grade I and the other third had that of grade IV (Figure 1).\n\n\nDiscussion\n\nThe effects of dehydration appear in many aspects of our lives. The National Academies of Sciences, Engineering, and Medicine of the United States of America has recommended a daily water intake of ≥3.7 liters for males and ≥2.7 liters for females. If we break it down into the number of glasses, it can be appreciated that our participants fall a bit short according to that recommendation14. Considering the capacity of each glass of about 250 mL, most of the individuals with back pain in our study reported drinking 1.3-2.3 liters (5–9 glasses) of water daily. This is similar to the results by Lindeman RD et al., in which the majority (71%) of the participants were found to be drinking at least six glasses of water daily25.\n\nDehydration has emerged as a separate risk factor for back pain and as mentioned above, disc degeneration may be the mechanism behind it6. Interestingly back pain is a problem that was encountered more than once a week (44.3%) in our participants, the majority of whom were aged 21 to 30 years (58.7%), this data mirrors another study that states that back pain is common in the adolescent population, it is recurrent, increases with age and usually does not diminish with time. Most of the time it is regarded as a usual life experience27. One possible reason may relate to the conclusions of Salminen JJ et al., they found a high risk (Relative risk: 16, Confidence interval: 95%) of recurrent back pain in individuals with disc degeneration compared to the ones without it, and this degeneration of the disc starts at around the age of 20, increasing through young adulthood28.\n\nWe have presented the results in accordance with the standard guidelines, with back pain of more than 30 days in the last 12 months labeled as chronic LBP, and pain up to seven days labeled as mild or no back pain23,24. While grading the severity of chronic back pain we found that around one-third of the population had chronic pain of grade I (35%) and the other third had that of grade IV (32%).\n\nIn previous studies, the frequency of lower back pain was between 13–35% in males, and 17–38% in females29–32. Moreover, female dominance is noted in this case overall33,34. Similarly, in this study, a slight female preponderance was noted. As explained in the literature, one cause of this might be a lower pain threshold in females, a better ability to perceive and discriminate the pain stimulus, poor tolerance to pain (as compared to males), or earlier onset of puberty35–37. Moreover, being female has been identified as an independent predisposing factor for dehydration38.\n\nWe have assessed the effects of back pain only in young adults and the difference in their daily activities. Further assessment should be performed in other age groups to evaluate the potential relationship of these activities with dehydration. Moreover, a comparative study across age groups, would allow the effect of aging to be determined. Our results cannot be generalized being collected from a single medical school and affiliated hospital, and from a single age group. We were also limited by the study design as the participants were not followed up to evaluate this back pain, especially the causes and outcomes.\n\n\nConclusion\n\nIn light of the results of the study, it can be concluded that with the increase in the pace of life many of the individuals who belong to the abovementioned age group have a reduced intake of water, and due to a probable relationship between LBP and dehydration, this might be a reason for the increasing propensity of LBP among them. There is an underlying need for further work in this regard.\n\nThe study was assessed and approved by the Institutional Review Board of Department of orthopedic surgery, Dow University of Health Sciences and Dr. Ruth Pfau Civil Hospital (Ortho/015/2019) Karachi, Pakistan. Written informed consent was obtained from all participants prior to participation.\n\n\nData availability\n\nFigshare: Characteristics of back pain in young adults and their relationship with dehydration. https://doi.org/10.6084/m9.figshare.11786457.v426\n\nThis project contains the following underlying data:\n\nUntitled1.sav (Participant responses to study questionnaire)\n\nFigshare: Characteristics of back pain in young adults and their relationship with dehydration. https://doi.org/10.6084/m9.figshare.11786457.v426\n\nThis project contains the following extended data:\n\nQuestionnaire Final.docx (Study questionnaire)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nKamper SJ, Henschke N, Hestbaek L, et al.: Musculoskeletal pain in children and adolescents. Braz J Phys Ther. 2016; 20(3): 275–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJones GT, Macfarlane GJ: Epidemiology of low back pain in children and adolescents. Arch Dis Child. 2005; 90(3): 312–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShiri R, Solovieva S, Husgafvel-Pursiainen K, et al.: Incidence of nonspecific and radiating low back pain: followup of 24-39-year-old adults of the Young Finns Study. Arthritis Care Res (Hoboken). 2010; 62(4): 455–9. PubMed Abstract \| Publisher Full Text\n\nAndersson GB: Epidemiological features of chronic low-back pain. Lancet. 1999; 354(9178): 581–5. PubMed Abstract \| Publisher Full Text\n\nBiering-Sørensen F, Kjøller M: [Musculoskeletal disorders]. Ugeskr Laeger. 2004; 166(14): 1331–3. PubMed Abstract\n\nKeating R: Can Dehydration Cause Back Pain? 2019. Reference Source\n\nMaughan RJ: Impact of mild dehydration on wellness and on exercise performance. Eur J Clin Nutr. 2003; 57(Suppl 2): S19–23. PubMed Abstract \| Publisher Full Text\n\nHendry NG: The hydration of the nucleus pulposus and its relation to intervertebral disc derangement. J Bone Joint Surg Br. 1958; 40-B(1): 132–44. PubMed Abstract \| Publisher Full Text\n\nGauchard GC, Gangloff P, Vouriot A, et al.: Effects of exercise-induced fatigue with and without hydration on static postural control in adult human subjects. Int J Neurosci. 2002; 112(10): 1191–206. PubMed Abstract \| Publisher Full Text\n\nPopkin BM, D’Anci KE, Rosenberg IH: Water, hydration, and health. Nutr Rev. 2010; 68(8): 439–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiem K, Lentner C, Seldrup J: Geigy scientific tables. 8th rev. a. Lentner C editor. Journal of Applied Toxicology. 1981; 7: 413. Reference Source\n\nMentes J: Oral hydration in older adults: greater awareness is needed in preventing, recognizing, and treating dehydration. Am J Nurs. 2006; 106(6): 40–9; quiz 50. PubMed Abstract \| Publisher Full Text\n\nEngell D, Hirsch E: Environmental and sensory modulation of fluid intake in humans. In: Ramsay DJ, Booth D, editors. Thirst, Physiological and Psychological Aspects. London: Springer; 1991; 382–390. Publisher Full Text\n\nSawka MN, Cheuvront SN, Carter R 3rd: Human Water Needs. Nutr Rev. 2005; 63(6 Pt 2): S30–9. PubMed Abstract \| Publisher Full Text\n\nThomas DR, Cote TR, Lawhorne L, et al.: Understanding Clinical Dehydration and Its Treatment. J Am Med Dir Assoc. 2008; 9(5): 292–301. PubMed Abstract \| Publisher Full Text\n\nGoldstein DJ: Beneficial health effects of modest weight loss. Int J Obes Relat Metab Disord. 1992; 16(6): 397–415. PubMed Abstract\n\nArmstrong LE, Costill DL, Fink WJ: Influence of diuretic-induced dehydration on competitive running performance. Med Sci Sports Exerc. 1985; 17(4): 456–61. PubMed Abstract \| Publisher Full Text\n\nGanesan S, Acharya AS, Chauhan R, et al.: Prevalence and Risk Factors for Low Back Pain in 1,355 Young Adults: A Cross-Sectional Study. Asian Spine J. 2017; 11(4): 610–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDietary Supplement Fact Sheet: Vitamin D. Fact Sheet for Health Professionals. Office of Dietary Supplements, National Institutes of Health. 2019. Reference Source\n\nHasan MM, Yaqoob U, Ali SS, et al.: Frequency of Musculoskeletal Pain and Associated Factors among Undergraduate Students. Case Reports Clin Med. 2018; 7(2): 131–45. Publisher Full Text\n\nKuorinka I, Jonsson B, Kilbom A, et al.: Standardised Nordic questionnaires for the analysis of musculoskeletal symptoms. Appl Ergon. 1987; 18(3): 233–7. PubMed Abstract \| Publisher Full Text\n\nVon Korff M, Ormel J, Keefe FJ, et al.: Grading the severity of chronic pain. Pain. 1992; 50(2): 133–49. PubMed Abstract \| Publisher Full Text\n\nDunn KM, Jordan K, Croft PR: Characterizing the course of low back pain: a latent class analysis. Am J Epidemiol. 2006; 163(8): 754–61. PubMed Abstract \| Publisher Full Text\n\nGriffith LE, Shannon HS, Wells RP, et al.: Individual participant data meta-analysis of mechanical workplace risk factors and low back pain. Am J Public Health. 2012; 102(2): 309–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLindeman RD, Romero LJ, Liang HC, et al.: Do elderly persons need to be encouraged to drink more fluids? J Gerontol A Biol Sci Med Sci. 2000; 55(7): M361–5. PubMed Abstract \| Publisher Full Text\n\nYaqoob U: Characteristics of back pain in young adults and their relationship with dehydration. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11786457.v4\n\nBurton KA, Clarke RD, McClune TD, et al.: The natural history of low back pain in adolescents. Spine (Phila Pa 1976). 1996; 21(20): 2323–8. PubMed Abstract \| Publisher Full Text\n\nSalminen JJ, Erkintalo MO, Pentti J, et al.: Recurrent low back pain and early disc degeneration in the young. Spine (Phila Pa 1976). 1999; 24(13): 1316–21. PubMed Abstract \| Publisher Full Text\n\nPietri F, Leclerc A, Boitel L, et al.: Low-back pain in commercial travelers. Scand J Work Environ Health. 1992; 18(1): 52–8. PubMed Abstract \| Publisher Full Text\n\nSmedley J, Egger P, Cooper C, et al.: Prospective cohort study of predictors of incident low back pain in nurses. BMJ. 1997; 314(7089): 1225–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEriksen W, Natvig B, Bruusgaard D: Smoking, heavy physical work and low back pain: a four-year prospective study. Occup Med (Lond). 1999; 49(3): 155–60. PubMed Abstract \| Publisher Full Text\n\nGyntelberg F: One year incidence of low back pain among male residents of Copenhagen aged 40-59. Dan Med Bull. 1974; 21(1): 30–6. PubMed Abstract\n\nKopec JA, Sayre EC, Esdaile JM: Predictors of back pain in a general population cohort. Spine (Phila Pa 1976). 2004; 29(1): 70–7; discussion 77–8. PubMed Abstract \| Publisher Full Text\n\nLake JK, Power C, Cole TJ: Back pain and obesity in the 1958 British birth cohort. cause or effect? J Clin Epidemiol. 2000; 53(3): 245–50. PubMed Abstract \| Publisher Full Text\n\nRiley JL 3rd, Robinson ME, Wise EA, et al.: Sex differences in the perception of noxious experimental stimuli: a meta-analysis. Pain. 1998; 74(2–3): 181–7. PubMed Abstract \| Publisher Full Text\n\nBerkley KJ: Sex Differences in Pain. Behav Brain Sci. 1997; 20(3): 371–80. PubMed Abstract \| Publisher Full Text\n\nJeffries LJ, Milanese SF, Grimmer-Somers KA: Epidemiology of adolescent spinal pain: a systematic overview of the research literature. Spine (Phila Pa 1976). 2007; 32(23): 2630–7. PubMed Abstract \| Publisher Full Text\n\nRowat A, Graham C, Dennis M: Dehydration in hospital-admitted stroke patients: detection, frequency, and association. Stroke. 2012; 43(3): 857–9. PubMed Abstract \| Publisher Full Text" }	[ { "id": "70056", "date": "07 Sep 2020", "name": "Rodrigo Meucci", "expertise": [ "Reviewer Expertise Epidemiology (musculoskeletal 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\nGeneral comment:\nThe objective of the manuscript was to establish whether there was a potential relationship between dehydration and low back pain among individuals aged 20 to 40 years in the general population. Unfortunately, the methods are not adequate to answer such a relevant scientific question.\n\nSpecific comments:\nThe exposure (dehydration), is inaccurate. The authors did not assess whether the respondents’ water consumption was adequate. Instead, they report only about the subjects’ self-perceived water intake.\n\nMedical students and young doctors are a specific group that do not represent the general population.\n\nThe authors did not apply an adequate data analysis and the results are poorly described. There is not even a p-value or a confidence interval. The authors did not mention adjustment for confounding factors.\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 source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "73379", "date": "16 Nov 2020", "name": "Aminu A Ibrahim", "expertise": [ "Reviewer Expertise Musculoskeletal physiotherapy", "Low back pain", "Knee pain", "Motor control exercise", "Patient education", "Questionnaire translation and validation", "Cross-cultural adaptation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me the opportunity to review this manuscript. The manuscript is very interesting for aiming to explore the potential relationship between dehydration and low back pain among young adults. The reviewer appreciates the excellent work of the authors. The manuscript is well-written and clear. The background, objectives, design, methods, statistics, and discussion are clearly explained and justified. However, I have some observations/corrections as appended below.\nAbstract:\nConclusion: \"…. who belong to the above mentioned age group….\" Please mention the age group.\n\nIntroduction:\n\"A study published in the International Journal of Neuroscience suggested that….\" Correct as: \"A previous research suggested that….\"\n\nMethods:\n\"…. tertiary care hospital from from March to May 2019.\" “from” is replicated, delete one.\n\n\"Proformas were distributed during lectures and workshops.\" Delete the sentence. It is a replica in the subsequent paragraph.\n\n\"Considering an estimated prevalence of LBP in young adults as 42.4%, confidence level of 95%, and a design effect of 1, the minimum sample size was kept as 376 and in the assigned time period of three months, a total sample of 426 was collected18.\"\nReference “18” was not placed correctly. Consider correcting as:\n\"Considering an estimated prevalence of LBP in young adults as 42.4%18, confidence level of 95%, and a design effect of 1, the minimum sample size was kept as 376 and in the assigned period of three months, a total sample of 426 was collected.\"\n\n\"….enzyme linked immunosorbent assay (ELISA)….\" Correct as: \"enzyme-linked immunosorbent assay (ELISA)\".\n\nResults:\n\"The authors also asked about the daily activities of the participants: 57.1% (n=206) reported…\" Correct as: \"Regarding the daily activities of the participants: 57.1% (n=206) reported…\"\n\n\"We applied the graded chronic pain scale to assess the severity of back pain where around one-third of the population….\" Correct as: \"Assessment of severity of back pain with graded chronic pain scale revealed that around one-third of the population….\"\n\nDiscussion:\n\"….according to that recommendation14.\" Correct as: ….according to this recommendation14.\n\n\"One possible reason may relate to the conclusions of Salminen JJ et al., they found a high risk (Relative risk: 16, Confidence interval: 95%) of recurrent back pain in individuals with disc degeneration compared to the ones without it, and this degeneration of the disc starts at around the age of 20, increasing through young adulthood28.\"\nThe above paragraph is not very clear. Please consider revising. Saminen JJ et al., should be written as \"Salminen et al.,\"\n\n\"….between 13–35% in males, and….\" Delete the comma (,) after males.\n\n….comparative study across age groups, would…. Delete the comma (,) after groups.\n\nThe discussion regarding your findings of the relationship between back pain and dehydration is scanty. I would like the author to discuss more on this.\n\nConclusions:\nThe subheading “Conclusion” should be written as “Conclusions”. Correct also for the abstract.\n\n\"…abovementioned age group…\" As mentioned earlier, write the age group.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7812", "date": "22 Feb 2022", "name": "Muniba Mehmood", "role": "Author Response", "response": "Thank you so much for giving us your valuable time and reviewing our manuscript. Following are the responses to your comments and a newer version has been uploaded after making changes according to your suggestions. Abstract: Conclusion: \"…. who belong to the above mentioned age group….\" Please mention the age group. DONE Introduction: \"A study published in the International Journal of Neuroscience suggested that….\" Correct as: \"A previous research suggested that….\" DONE Methods: \"…. tertiary care hospital from from March to May 2019.\" “from” is replicated, delete one. DONE \"Proformas were distributed during lectures and workshops.\" Delete the sentence. It is a replica in the subsequent paragraph. DONE \"Considering an estimated prevalence of LBP in young adults as 42.4%, confidence level of 95%, and a design effect of 1, the minimum sample size was kept as 376 and in the assigned time period of three months, a total sample of 426 was collected18.\" Reference “18” was not placed correctly. Consider correcting as: \"Considering an estimated prevalence of LBP in young adults as 42.4%18, confidence level of 95%, and a design effect of 1, the minimum sample size was kept as 376 and in the assigned period of three months, a total sample of 426 was collected.\" DONE \"….enzyme linked immunosorbent assay (ELISA)….\" Correct as: \"enzyme-linked immunosorbent assay (ELISA)\". DONE Results: \"The authors also asked about the daily activities of the participants: 57.1% (n=206) reported…\" Correct as: \"Regarding the daily activities of the participants: 57.1% (n=206) reported…\" DONE \"We applied the graded chronic pain scale to assess the severity of back pain where around one-third of the population….\" Correct as: \"Assessment of severity of back pain with graded chronic pain scale revealed that around one-third of the population….\" DONE Discussion: \"….according to that recommendation14.\" Correct as: ….according to this recommendation14. DONE \"One possible reason may relate to the conclusions of Salminen JJ et al., they found a high risk (Relative risk: 16, Confidence interval: 95%) of recurrent back pain in individuals with disc degeneration compared to the ones without it, and this degeneration of the disc starts at around the age of 20, increasing through young adulthood28.\" The above paragraph is not very clear. Please consider revising. Saminen JJ et al., should be written as \"Salminen et al.,\" DONE \"….between 13–35% in males, and….\" Delete the comma (,) after males. DONE ….comparative study across age groups, would…. Delete the comma (,) after groups. DONE The discussion regarding your findings of the relationship between back pain and dehydration is scanty. I would like the author to discuss more on this. A PARAGRAPH HAS BEEN ADDED Conclusions: The subheading “Conclusion” should be written as “Conclusions”. Correct also for the abstract. DONE \"…abovementioned age group…\" As mentioned earlier, write the age group. DONE" } ] } ]	1	https://f1000research.com/articles/9-159
https://f1000research.com/articles/11-208/v1	18 Feb 22	{ "type": "Research Article", "title": "Impact of antiretroviral therapy regimens adherence on perceived health and wellbeing status among adults living with HIV in Ghana", "authors": [ "Awolu Adam", "Adam Fusheini", "FAITH A AGBOZO", "GEOFFREY ADEBAYO ASALU", "MARTIN AMOGRE AYANORE", "NORBERT AMUNA", "PRINCE KUBI APPIAH", "SENAM ABENA KLOMEGAH", "FRANCIS BRUNO ZOTOR", "Adam Fusheini", "FAITH A AGBOZO", "GEOFFREY ADEBAYO ASALU", "MARTIN AMOGRE AYANORE", "NORBERT AMUNA", "PRINCE KUBI APPIAH", "SENAM ABENA KLOMEGAH", "FRANCIS BRUNO ZOTOR" ], "abstract": "Introduction The efficacy of antiretroviral medicines to improve health outcomes and wellbeing depends largely on how soon they are prescribed and how PLHIV adhere to their prescribed regimens. The objectives of this cross-sectional study were to examine adherence to antiretroviral therapy (ART) and to assess perceived health and wellbeing among people living with HIV(PLHIV) in two regions in Ghana. Methods Quantitative data were collected from 301 PLHIV at three HIV clinics in Volta and Oti regions in Ghana using descriptive cross-sectional study design. Data collected included sociodemographic characteristics, ART initiation and adherence, and perception of health and wellbeing and analyzed using Statistical Package for Social Sciences (SPSS) version 20. Demographic and categorical variables were analyzed using descriptive statistics while Chi-squared analyses and binary logistics regression were used to determine variables that correlated with adherence to ART regimen. A Mann–Whitney U-test was used to measure differences in perception of health and wellbeing between male and female participants. Results 80% initiated ART within a month following diagnosis and 97.3% reported adhering to their ART regimens consistently. A Pearson χ2 revealed that the availability of alternatives to the current ART medications showed a significant association with ART adherence (χ2 =12.078, p= 0.002). 90% reported improvement in their health and personal wellbeing. High ART adherence and positive perceived health and wellbeing were found. Conclusion Regular supply of ART and investment in LA-ART to ensure more effective, efficient, and stress-free adherence to ART for PLHIV regardless of their residence in the country.", "keywords": [ "Antiretroviral", "adherence", "HIV", "PLWHA", "Ghana", "wellbeing" ], "content": "Introduction\n\nHIV remains a major global public health issue since its emergence, having claimed an estimated 36.3 million (27.2–47.8 million) lives and over 38 million people living with HIV (PLHIV) by the end of 2020 (World Health Organization, 2021). Of these numbers, over two-thirds (25.4 million) are in the WHO African Region (World Health Organization, 2021). In Ghana, there were an estimated 342,307 PLHIV (36% male, 64% female) by the end of 2019, with 20,068 new infections, and 13,616 AIDS-related deaths (Ghana AIDS Commission, 2019).\n\nDue to significant advancement in the development and access to more effective therapeutics, improvement in the care and management of PLHIV today is different from three decades ago. WHO reported that in 2020, 27.5 million (26.5–27.7 million) PLHIV were receiving antiretroviral therapy (ART). This equates to a global ART coverage rate of 73% (56–88%) (World Health Organization, 2021). This has led to an almost 59% suppression of the virus with minimal risk of infecting others in 2019 (UNAIDS, 2020). In Ghana, close to 68% of PLHIV receiving ART have achieved viral suppression (Ghana AIDS Commission, 2016). Between 2000 and 2019, new HIV infections fell by 39% and HIV-related deaths fell by 51%, with 15.3 million lives saved due to ART (World Health Organization). These improvements have been attributable to increased concerted efforts of national interventional programs and international funding. Thus, this indicates a remarkable improvement in the health and longevity of HIV-diagnosed persons compared with the 1980s and early 1990s.\n\nImprovements in ARTs, counseling and other HIV/AIDS care have been game-changers in the fight against the HIV pandemic. There is sufficient evidence that ART adherence and maintenance of viral suppression improve the health of PLHIV who maintain viral suppression; however, viral suppression also prevents infection from positive partners to negative sexual partners (World Health Organization, 2018). However, for ART to lead to viral suppression and overall improvement in health and quality of life for PLHIV, there must be sustained adherence to ART regimens; be it first- or second-line prescribed treatment. This is because adherence to the prescribed treatment regimen improves clinical outcomes and reduces chances of mortality (World Health Organization, 2018) while non-adherence is associated with poor health outcomes, frequent hospitalizations, and increased rates of mortality (Vrijens et al., 2012). Promoting ART adherence has, therefore, become one of the main goals of clinicians and professionals who are directly or indirectly caring for PLHIV (DiMatteo, 2004). With consistent adherence to ART, economic wellbeing improved significantly among adults in study participants in South Africa. This reduced their inability to perform required job activities from 56% to 6% and reduced dependence on caretakers from 81% to less than 1% over a five-year period (Rosen et al., 2014). Also, among other factors, poor ART adherence has been linked to malnutrition, under-nutrition, and food insecurity among PLHIV in different settings (Gebremichael et al., 2018; Negessie et al., 2019; Weldehaweria et al., 2017). On the other hand, non-adherence is associated with poor health outcomes, frequent hospitalizations, and increased rates of mortality (DiMatteo, 2004). Promoting ART adherence has, therefore, become one of the main goals of clinicians and professionals who are directly or indirectly caring for PLHIV (Weiser et al., 2017).\n\nMany studies have been conducted to evaluate ART adherence in various countries and there have been mixed results. While some studies reported good adherence, others report poor adherence. For instance, it was reported that only 55% of PLHIV globally adhere to ART indicating a poor global adherence index (Saberi et al., 2015). Poor ART adherence, ranging between 30% and 50% has also been reported in the USA (Marcum et al., 2013). Dalmida and colleagues indicated that among adults in the US receiving ART, only 25% have viral suppression, which suggests that poor ART adherence may partly be to blame for the low viral suppression (Dalmida et al., 2017). However, Kim et al. and Yu et al. have reported 70.4% and 86% adherence rates among PLHIV in Korea and China respectively (Kim et al., 2018; Yu et al., 2018).\n\nIn Ghana, the Ghana Aids Commission (GAC) reported a 77% sustained ART adherence among PLHIV leading to 68% viral suppression (GAC, 2019). Lokpo et al., and Afrane et al. have also reported 69% and 61.6% viral suppression among adults and children respectively in two cities in Ghana (Afrane et al., 2021; Lokpo et al., 2020). However, other studies in Ghana have reported lower ART adherence among various groups of PLHIV in the country (Abrokwah, 2018; Anokye-Kumatia et al., 2018; Nichols et al., 2019).\n\nVarying factors have been reported for adherence and non-adherence of ART among PLHIV in different parts of the world. The most common factors reported for non-adherence to ART included fear of stigma, gender, non-disclosure of HIV status, younger age, and depression among PLHIV (Kim et al., 2018; Madiba & Josiah, 2019; Yu et al., 2018). Other predictors of non-adherence reported include race, alcohol use, and low socio-economic status (SES) (Amirkhanian et al., 2018; Corless et al., 2017). In Ghana, similar factors, such as gender, age, and fear of stigma in addition to financial difficulties, clinic attendance, educational status, and shortage of antiretroviral medications have also been reported to predict ART adherence (Anokye-Kumatia et al., 2018; Nichols et al., 2019; Poku et al., 2020).\n\nIn recent years, there has been an increasing interest in ART adherence and the issues of quality of life and wellbeing among PLHIV (Ghiasvand et al., 2019). However, in Ghana, and the Volta and Oti regions, in particular, there has been little quantitative analysis of ART adherence and perception of wellbeing among PLHIV. This study sets out to assess the self-reported adherence of ART and perceptions of wellbeing among PLHIV receiving ART at three HIV clinics in the Volta and Oti regions of Ghana.\n\n\nMethods\n\nThis study is part of a broader HIV/AIDS study on stigmatization and discrimination among PLHIV in Ghana. The first part of the study, which focused on HIV stigma and status disclosure in three Municipalities in Ghana has been published (Adam et al., 2021). A descriptive cross-sectional study was employed to collect data from a sample of 301 PLHIV at HIV/AIDS clinics in Hohoe, Kpando, and Dodi Papase municipalities in the Volta and Oti regions.\n\nThe sample size calculation was based on the PLHIV population approximation of 2,500 at the study sites based on registered number of patients at the clinics. This sample size was derived using the sample determination formula by Kothari and Nachmias (Frankfort-Nachmias & Nachmias, 2007; Kothari, 2004). The formula recommended a sample size of 384 people, which we further increased by 10% to 423 to account for non-response rate. However, due to non-response and missing data, 301 (73.2%) was used in the analysis. Convenience sampling was used to sample participants into the study. The inclusion criteria for the study were being diagnosed with HIV, registered at the one of the clinics, receiving ART, and aged from 18 years and above.\n\nThe target population was PLHIV registered and receiving ART treatment in these three municipalities. The inclusion criteria were that a participant had to be clinically diagnosed with HIV and receiving ART at the HIV clinics. We used a semi-structured questionnaire to collect data from the participants. The questionnaire was written in simple and in lower-grade English language for easy understanding and response and administered by trained research assistants. Many of the data collectors were native speakers of Ewe, the predominant language in the study areas, and Akan is also commonly spoken. For those participants who did not understand the English language, translation was provided into Ewe and Akan. The questionnaire was pretested with some HIV-positive patients at the Hohoe municipal hospital and corrections were made accordingly. Those who participated in the pretest were not included in the actual data collection.\n\nData collected included socio-demographics, HIV status disclosure, factors for disclosure and nondisclosure, HIV stigma, ART adherence, and associated factors, and perception of wellbeing. Two outcome variables measured in this study were ART adherence and perception of personal wellbeing. ART adherence referred to the consistency of taking prescribed medication according to doctors’ recommendation while perception of personal welling is how the participant assesses his or her physical health, social, and economic wellbeing. The data were analyzed and presented using a statistical package for social science (SPSS) version 20. Categorical variables were reported as frequencies, percentages, and a pie chart. A Chi-squared (χ2) analysis and a binary logistic regression model was used to determine factors correlated with ART adherence, HIV status disclosure and internalized HIV-related stigma. A Mann–Whitney U-test was used to measure the difference in perception of wellbeing between male and female participants. The confidence level for all statistical analyses was set at 95% (0.05).\n\nThis study strictly adhered to the required ethical standards and procedures. The study received ethical approval from the Ghana Health Service Ethics Review Committee (GHSERC) with approval number GHSERC09/04/17. Permission was also received from the management of all the three health facilities involved in the study. The study participants were provided with an information sheet that explained the details of the study. This was to ensure that potential participants clearly understood the purpose, nature, and procedures involved in the study. The study adhered to the ethical principles of privacy and confidentiality of study participants as well as voluntary participation and withdrawal without any penalty. Above all, participants signed or thumb printed their signature before being recruited to be part of the study.\n\n\nFindings\n\nFrom Table 1, 244 (74.4%) of the study participants were female and only 77 (25.6%) were males. The majority, 139 (46.1%), of the participants were married and lived with their partners, 71 (23.5%) had never married and 91 (30.1%) were either separated or divorced. For educational attainment, 270 (89.7%) of the participants attained at least primary education. More than half, 160 (53.1%), attained junior high school (JHS) while 31 (10.2%) had no formal education. The majority of the participants were between the ages of 30 and 59 with 74 (24.5%), 98 (32.5%), and 61 (20.2%) being between ages 30–39, 40–49, and 50–59 respectively. 29 (9.6%) of the participants were under age 30. Other important demographic characteristics are presented in the table.\n\nThe time of initiation of ART is critical for the management of HIV including viral suppression and living a healthy life. The majority (181 (60%)) reported that they started ART immediately after they were tested and diagnosed of being HIV positive. We also found that 59 (19.6%), 44 (14.6%), and 17 (5.6%) started their ART within a month, within four months, and after one year of HIV diagnosis, respectively. Most (293 (97.3%)) of the participants reported that they take their prescribed medication consistently according to their ART regimen and only eight (2.7%) did not take their medication regularly. However, 76 (25.2%) reported they ever missed taking their medication with 44 (57%) of them missing their medication in the last four weeks before the study. The predominant reason given for missing their medication was forgetfulness (10.6%). The results are summarized in Table 2.\n\nWith the high rate of ART adherence, we wanted to determine the factors that had significant associations with ART. We computed the χ2 test of independence with various variables including socio-demographic variables, HIV status disclosure, self-internalized HIV, and social support among others. A Pearson χ2 revealed that only the availability of an alternative to the current ART medications showed a significant association with ART adherence in this study sample (χ2=12.078, p=0.002). In cross tabulation, those who took their anti-retroviral medication because they did not have an alternative were more likely to miss taking their medication compared to those who were satisfied with the current ART regimen for their HIV management. However, in a logistic regression model, none of the predictor variables significantly predicted ART adherence as shown in Table 3.\n\na Variable(s) entered on step 1: Sex of participant, Marital status, Educational status, Ethnic background, Religious affiliation, Level of internalized stigma, SSReceived, Current employment status, Alternative treatment.\n\nTen Likert questions were included in the questionnaire to assess the perception of health status and wellbeing among the participants following ART. For this analysis, four response categories for each of the questions (strongly agree, agree, disagree, and strongly disagree) were collapsed into agreeing and disagreeing. The questions covered access to HIV medication, effects of the medication, and general satisfaction of health and personal wellbeing. The results are presented in Table 4. ART is supposed to be free for all PLHIV who are attending the HIV clinics. Most (273 (91%)) reported that they receive their medication regularly and 251 (83.4%) do not consider their medication to be too many. For general health effects, 282 (94%), 274 (91%), and 250 (83%) felt that their ART helped them to eat well, gave them energy for their daily activities, and helped them to sleep well, respectively. On personal wellbeing, the majority (225 (75%)) reported that with ART, their HIV status did not limit their ability to work while 213 (71%) indicated that they were employed at the time of this study. Overall, 270 (90%) reported that their ART participation made them feel happy about themselves.\n\nA Mann–Whitney’s U-test was conducted to determine the difference in the overall happiness about the wellbeing between female and male participants in this study. A Mann–Whitney U-test revealed that female participants scored a higher mean happiness rank of 159.17 than males’ mean rank of 127.23, and this was statistically significant (U=6793.500, female=159.17, male=127.23, p=0.002). This implies that female PLHIV were more likely to report a higher personal wellbeing than male PLHIV participants on ART.\n\nParticipants were asked about challenges they encounter in their adherence to ART regimens as prescribed by their doctors. The majority (162 (53.8%)) indicated that they did not have a challenge in accessing and taking their medication. However, 106 (35.2%) reported that they have transportation challenge in traveling to and from their respective clinics for their medication. Other challenges included medication side effects (12 (4%)), medication shortage (11 (3.7%)), too many medications (seven (2.3%)), and cost of medication (three (1%)). These are presented in Figure 1.\n\n\nDiscussion\n\nThe initiation of ART among participants in this study was in line with the WHO 2016 consolidated guidelines on ART that recommends early initiation of ART for adults with HIV and CD4 count below 500 cells/mm2, regardless of the clinical stage to optimize positive HIV treatment outcomes and prevent new infections (World Health Organization, 2016). However, not many findings have reported early initiation of ART especially in developing and resource-limited countries. Meanwhile, in this study, 60%, 19.6%, and 14.6% of participants reported that they initiated ART immediately after their diagnosis, within a month, and within four months, respectively. This indicates that about 95% of the participants in this study enrolled in ART within six months after their HIV diagnosis which is consistent but higher than 53% reported in a systematic review in Australia in 2015 (McManus et al., 2019). In an analysis of a population-based sample in San Francisco to track early initiation of ART, researchers reported a steady increase from early ART initiation from 2001 to 2015 which peaked at 74% (Truong et al., 2019).\n\nIt was also found in this study that ART adherence was among the highest reported in the literature as reported by the participants in the study. A 55% global adherence rate of ART was reported a few years earlier among PLHIV (Saberi et al., 2015). In this study, most (97%) of the participants reported that they adhere to ART regimens and are consistent with their anti-retroviral medications since initiating ART. This adherence rate is higher than the 78% national adherence rate reported by the Ghana AIDS Commission in Ghana (Ghana AIDS Commission, 2019). The high adherence rate is also consistent with but higher than 70.4% and 86% reported in Korea and China, respectively (Kim et al., 2018; Yu et al., 2018). Again, the finding is higher than the 30–50% ART adherence rates reported in the USA earlier (Marcum et al., 2013).\n\nMany factors have been reported to predict or at least to be associated with ART adherence among PLHIV in different environments. In this study, different variables were tested using different statistics and only the availability of alternatives to the current ART had a statistically significant association with ART adherence in χ2 analysis. Those who took their medication because they did not have alternatives to the current ART regimen were more likely not to adhere to ART compared to those who did not worry about alternatives to the current ART regimen. However, in binary and multinomial regression analysis the same variable did show a significant relationship to ART adherence. This finding is inconsistent with findings in Ghana where gender, fear of stigma, shortage of antiretroviral drugs, and financial difficulties were reported to predict ART adherence (Anokye-Kumatia et al., 2018; Nichols et al., 2019; Poku et al., 2020). Other promoters of ART adherence reported in a teaching hospital in Accra, Ghana; included perception of benefits of ART, awareness of ART regimen, access to food, and transparency on the part of clinicians (Dzansi et al., 2020), which we did not find in this study. The finding is also inconsistent with findings of fear of HIV stigma, gender, and non-disclosure of HIV status, and lower SES (Corless et al., 2017; Kim et al., 2018; Madiba & Josiah, 2019; Yu et al., 2018).\n\nThe finding is, however, consistent with emerging evidence in the literature that PLHIV around the world are searching and showing a preference for alternative ARTs and willing to switch from the existing daily pills regimen to long-acting ARTs (LA-ART) including six months injectables, implants, and ART-free HIV remission (Dandachi et al., 2021). For example, in a study to explore the perceptions and viewpoints of PLHIV about LA-ART in France, researchers concluded that LA-ART may be the suitable mode of HIV treatment for some PLHIV (Carillon et al., 2020). In a quantitative study among 282 PLHIV in the USA, Dubé and colleagues reported that 42% and 24% indicated their preference of six-month injectables and ART-free HIV remission respectively over the daily pills (Dubé et al., 2020). Similarly, in Texas, researchers reported that 61% of study participants preferred LA-ART over the daily pills regimen and specifically, 41%, 40%, and 18% preferred a pill for six months, injectables, and implants respectively (Dandachi et al., 2021).\n\nTherefore, the finding in this study may be a signal that alternatives to the current ART regimens in Ghana should be taken into consideration by the Ghana AIDS Commission, Ghana Health Service, and Ghana AIDS Control program in planning and updating HIV/AIDS policies in Ghana.\n\nMany health and social benefits of ART adherence have been reported for those who have consistently adhered to ART regimen; including viral suppression and reduced risk of infecting others, improved quality of life, and reduction in AIDS-related deaths. There seems to be a consensus in the literature that ART adherence has greater health benefits for PHLIV. For instance, the WHO reported that 67% of all those estimated to be living with HIV globally were receiving ART out of which 59% achieved suppression of the HIV with no risk of infecting others (UNAIDS, 2020; World Health Organization, 2021). The Ghana AIDS Commission and Lokpo et al. have also reported 68% and 61.6% suppression among different PLHIV groups in Ghana who have sustained ART adherence (Ghana AIDS Commission, 2019; Lokpo et al., 2020). This study was not a clinical one and so viral suppression and CD4+ count were not measured to determine the health outcomes for the participants. However, self-reported health status and wellbeing were assessed whereby 90% of the participants reported being happy with their overall health and personal wellbeing. Specifically, 94%, 91%, 83%, and 75% of the participants rated their nutritional status, energy level, sleep behavior, and ability to work, respectively, significantly improved while on ART. The findings of improvement in personal wellbeing among PHLIV on ART are corroborated by the findings of other studies that show that data from various sources show improved quality of life especially when ART is initiated early such as reported in this study (Ford et al., 2018; Lifson et al., 2017). The risk of early death due to HIV-related causes has also been found to be significantly reduced with early initiation and adherence to ART (Mangal et al., 2019). The findings of improvement in physical, economic, and general wellbeing are also consistent with findings from similar studies. For instance, in a study of perceptions of the benefits of ART among PLHIV in Cote d’Ivoire, Brazil, and Ethiopia, researchers found that participants adhered to ART regimen because they perceived ART to reduce the risk of transmission of HIV, minimize job losses, and improves economic productivity among other benefits (Abebe Weldsilase et al., 2018; Dutra et al., 2019; Hendrickson et al., 2019). These findings imply that the more PLHIV perceive that adhering to ARTs has direct personal health and economic benefits, the more likely they will be consistent in their adherence to ARTs.\n\nThe findings in this study are based on self-reported data. We encourage caution in drawing further conclusions as the researchers could not substantiate the self-reported information with clinical parameters. We also want to caution that since only the availability of ART alternative had significant association with ART adherence in χ2 analysis but was nonsignificant in logistic regression, predicting adherence with that variable is inconclusive. However, achieving 73% of the targeted sample size with a sub-population often difficult to reach due to fear of stigma was a strength worth noting.\n\n\nConclusion\n\nThe findings from this study showed a high level of ART adherence among PLHIV in the three localities covered and corroborates reports of high and consistent ART adherence by the Ghana AIDS Commission. We also found in this study that availability and accessibility to alternatives to the current ART regimen may be a strong motivator for adherence. Finally, our findings point that initiation and adherence to ART could lead to improvement in health and wellbeing of PLHIV as demonstrated by the self-report of participants. For public health practice, we advocate continuous effort to motivate PLHIV to adhere to their ART regimen through enhanced access to a regular supply of ART. Investment in LA-ART to ensure more effective, efficient, and stress-free adherence to ART for all PLHIV need to be expedited.\n\n\nData availability\n\nThe data from which the results and conclusions of this manuscript are drawn came from PLHIV who are considered a vulnerable population. As such, every effort was made during the collection, handling, and analysis to protect the personal information and health records of the participants. To continue to ensure that we adhere to the principles of data privacy and confidentiality, we have created a limited dataset which is de-identified and have deposited it in Zenodo with the DOI: 10.5281/zenodo.5914548. A request to the principal investigator or the Head of the Department of Family and Community Health is still required for full access and use of the data.", "appendix": "Acknowledgements\n\nThe workload involved in the conduct of the study was such that without the support of people at different stages and collectively, we would encountered more challenges. A number of important individuals provided critical support for the researchers from start to the end of the study and we would like to acknowledge their immense contributions in conducting the study. The coordinators of the HIV clinics at Hohoe Municipal Hospital, Margarete Marquart Hospital in Kpando, and St. Mary Theresa Hospital at Dodi Papase coordinated the informed consent process, securing permission to conduct the study at their facilities, and supported our data collectors. We also gratefully acknowledge the support of selected students of the School of Public Health who volunteered to be trained as data collectors and who helped significantly in the data collection for the study. We sincerely thank all others who helped in diverse ways to make the study a success.\n\n\nReferences\n\nAbebe Weldsilase Y, Likka MH, Wakayo T, et al.: Health-related quality of life and associated factors among women on antiretroviral therapy in health facilities of Jimma Town, Southwest Ethiopia. Adv. Public Health. 2018; 2018: 1–12. Publisher Full Text\n\nAbrokwah BJ: Factors associated with Antiretroviral Therapy adherence amongst men who have sex with men in selected facilities. University of Ghana; 2018.\n\nAdam A, Fusheini A, Ayanore MA, et al.: HIV Stigma and Status Disclosure in Three Municipalities in Ghana. Ann. Glob. Health. 2021; 87(1): 49. 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Publisher Full Text\n\nHendrickson ZM, Naugle DA, Tibbels N, et al.: “You Take Medications, You Live Normally”: The Role of Antiretroviral Therapy in Mitigating Men’s Perceived Threats of HIV in Côte d’Ivoire. AIDS Behav. 2019; 23(9): 2600–2609. PubMed Abstract \| Publisher Full Text\n\nKim J, Lee E, Park B-J, et al.: Adherence to antiretroviral therapy and factors affecting low medication adherence among incident HIV-infected individuals during 2009–2016: a nationwide study. Sci. Rep. 2018; 8(1): 1–8. Publisher Full Text\n\nKothari CR: Research methodology: Methods and techniques. New Age International; 2004.\n\nLifson AR, Grund B, Gardner EM, et al.: Improved quality of life with immediate versus deferred initiation of antiretroviral therapy in early asymptomatic HIV infection. AIDS (London, England). 2017; 31(7): 953–963. PubMed Abstract \| Publisher Full Text\n\nLokpo SY, Ofori-Attah PJ, Ameke LS, et al.: Viral Suppression and Its Associated Factors in HIV Patients on Highly Active Antiretroviral Therapy (HAART): A Retrospective Study in the Ho Municipality, Ghana. AIDS Res. Treat. 2020; 2020: 1–7. Publisher Full Text\n\nMadiba S, Josiah U: Perceived stigma and fear of unintended disclosure are barriers in medication adherence in adolescents with perinatal HIV in Botswana: A qualitative study. Biomed Res. Int. 2019; 2019: 1–9. PubMed Abstract \| Publisher Full Text\n\nMangal TD, Meireles MV, Pascom ARP, et al.: Determinants of survival of people living with HIV/AIDS on antiretroviral therapy in Brazil 2006–2015. BMC Infect. Dis. 2019; 19(1): 1–9. Publisher Full Text\n\nMarcum ZA, Sevick MA, Handler SM: Medication nonadherence: a diagnosable and treatable medical condition. JAMA. 2013; 309(20): 2105–2106. 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PubMed Abstract \| Publisher Full Text\n\nRosen S, Larson B, Rohr J, et al.: Effect of antiretroviral therapy on patients’ economic well being: five-year follow-up. AIDS (London, England). 2014; 28(3): 417–424. PubMed Abstract \| Publisher Full Text\n\nSaberi P, Neilands TB, Vittinghoff E, et al.: Barriers to antiretroviral therapy adherence and plasma HIV RNA suppression among AIDS clinical trials group study participants. AIDS Patient Care STDs. 2015; 29(3): 111–116. PubMed Abstract \| Publisher Full Text\n\nTruong H-HM, Pipkin S, Grant RM, et al.: Increased uptake of early initiation of antiretroviral therapy and baseline drug resistance testing in San Francisco between 2001 and 2015. PLoS One. 2019; 14(3): e0213167. PubMed Abstract \| Publisher Full Text\n\nUNAIDS: Graph of the week, 90–90–90: good progress, but the world is off-track for hitting the 2020 targets. UNAIDS; 2020.\n\nVrijens B, De Geest S, Hughes DA, et al.: A new taxonomy for describing and defining adherence to medications. Br. J. Clin. Pharmacol. 2012; 73(5): 691–705. PubMed Abstract \| Publisher Full Text\n\nWeiser J, Beer L, Brooks JT, et al.: Delivery of HIV antiretroviral therapy adherence support services by HIV care providers in the United States, 2013 to 2014. Journal of the International Association of Providers of AIDS Care (JIAPAC). 2017; 16(6): 624–631. PubMed Abstract \| Publisher Full Text\n\nWeldehaweria NB, Abreha EH, Weldu MG, et al.: Psychosocial correlates of nutritional status among people living with HIV on antiretroviral therapy: A matched case-control study in Central zone of Tigray, Northern Ethiopia. PloS one. 2017; 12(3): e0174082. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Research & development for HIV/AIDS, Global Strategic Direction.\n\nWorld Health Organization: Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach. World Health Organization; 2016.\n\nWorld Health Organization: Viral suppression for HIV treatment success and prevention of sexual transmission of HIV. World Health Organization; 2018.\n\nWorld Health Organization: HIV/AIDS Key Facts. World Health Organization; 2021.\n\nYu Y, Luo D, Chen X, et al.: Medication adherence to antiretroviral therapy among newly treated people living with HIV. BMC Public Health. 2018; 18(1): 1–8. Publisher Full Text" }	[ { "id": "151742", "date": "24 Oct 2022", "name": "Raphael Z. Sangeda", "expertise": [ "Reviewer Expertise ART adherence", "Antimicrobial resistance", "HIV drug resistance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of the article \"Impact of antiretroviral therapy regimens adherence on perceived health and well-being status among adults living with HIV in Ghana\" try to examine adherence to antiretroviral therapy (ART) and the perceived well-being of people living with HIV in two regions of Ghana.\nIn my view, the issue of adherence to ART is critical and requires a thorough investigation from time to time. However, the authors must address specific issues to improve the article's readability.\nTitle\nI suggest the title: \"Impact of perceived health and well-being on antiretroviral therapy regimens adherence status among adults living with HIV in Ghana\".\n\nAbstract\nDefine abbreviations such as PLHIV on first use.\n\nThe methods section mentioning the use of descriptive statistics for demographic and categorical data can be amended for accuracy.\n\nPlease mention in the methods section of the abstract how adherence was measured (questionnaire and how it was scored).\n\nThe results section opening word cannot be a number; change the writing style.\n\nThe word LA-ART is not defined in the conclusion of the abstract.\n\nAt the end of the abstract, mention the country you are referring to.\n\nIntroduction\nIn the second paragraph of the introduction, therapeutics could be changed to ART, and more use of recent references when citing literature.\n\nThe word ART and other abbreviations should be spelled out on first use.\n\nThe word HIV/AIDS can be changed to HIV to make it more appropriate for the condition in the era of test and treatment.\n\nIn the third paragraph of the introduction, avoid reparations of ideas. Also, make it clear why counseling is part of this writing.\n\nIn the fifth paragraph, delete the words \"in the country.\"\n\nMethods\nIn the data collection and analysis section, participants can choose to be interviewed in English or Akan. This language use can introduce bias in your collected data.\n\nIn the fourth paragraph, there is a lack of details on tools used to measure and score adherence.\n\nResults\nIn Table 1, the total number (N) for each variable is unclear. Add N to each variable category or the table caption, or both.\n\nPage 6: The second paragraph reports \"high adherence.\" Where is this cited? Which Table\n\nTable 3 can be simplified and presented without the statistical software details.\n\nFigure 1: No values are labeled on each slice of the pie chart.\n\nThe first paragraph on page 8: How was \"happiness\" measured? Add to the methods before using the data.\n\nThe results showing the impact of regimens/therapies used are missing in this section even though the title and conclusions state the impact of regimens; show the impact of regimens and discuss it.\n\nDiscussion\nIn the first paragraph of the discussion, the CD4 criteria are no longer used in the test and treatment area.\n\nIn the second paragraph, be cautious that comparing adherence depends on the method used to test compliance.\n\nWhich method was used to measure and score adherence in this study?\n\nAlso, note that adherence assessment in the past years may not be fairly compared with current rates, given the changes in regimens that have taken place over the years.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "157221", "date": "05 Dec 2022", "name": "Esmaeil Mehraeen", "expertise": [ "Reviewer Expertise · Health information technology· Mobile Health· Health informatics· Data mining· Artificial intelligence· Infectious diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is clear and well organized. However, I may require some comments on the following issues.\nAbstract: This section was well-written and easy to understand. In addition, the objective of the study and the state of the art of the study are clear. Furthermore, the keywords should be represented the study. Please match the keywords with the mesh.\nIntroduction: Based on my review, I recommend that the authors should add various improvements, such as: - Hypothesis and objectives. - Add a statement or sentence about whether there are any similar studies that have been done before. - Add the important issues.\nMaterial and Methods: This section was well-written\nConclusion: In this part of the manuscript, the authors have been successfully established the conclusion of this research. However, it might be useful if the authors mention the suggestion for forthcoming research.\nReferences: The core references are acceptable, but I think the authors should add the latest references in this manuscript. Please mentioned many high-quality COVID-19 related papers, such as1,2,3.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-208
https://f1000research.com/articles/11-207/v1	18 Feb 22	{ "type": "Study Protocol", "title": "A new minimally invasive, non-excisional, surgical browlift technique with minimal scarring: a protocol for a prospective observational study", "authors": [ "Frank W. de Jongh", "Laurens B.R. Kooiman", "Elijah E. Sanches", "Sjaak Pouwels", "Koen J.A.O. Ingels", "Kim M.E. Wehrens", "Liang T. Tan", "Frank W. de Jongh", "Laurens B.R. Kooiman", "Elijah E. Sanches", "Koen J.A.O. Ingels", "Kim M.E. Wehrens", "Liang T. Tan" ], "abstract": "Background The aim of this study is to prospectively evaluate the new minimal invasive (MINE) browlift technique with possibly superior results and minimal visible scarring. Methods A prospective observational study will be performed on all available data from patients who will undergo a browlift procedure in the Haaglanden Medical Center from 1-6-2021 till 31-5-2022. Our goal is to include at least 50 patients (1 per week). Inclusion criteria are: patients with medical (i.e. brow-ptosis, facial paralysis) or cosmetic indication, patients with sufficient understanding of the Dutch or English language and willingness to participate in extra study specific follow-up moments and filling in of questionnaires. Exclusion criteria are: <18 years of age and patients with previous brow or eyelid surgery. Patients will be photographed pre- and postoperatively using the VECTRA camera. The database management software Castor will be used to store and collect the data from the questionnaire. The Medical Research Ethics Committee found this study not eligible to be submitted to the Dutch Medical Research Involving Human Subjects Acts (WMO). Written informed consent will be obtained from all patients. Results Outcomes measures to be evaluated include: scarring after procedure; functionality of eyebrow movement; amount of correction in brow ptosis, measured in VECTRA; longevity of procedure in months; aesthetic result as assessed by questionnaires; and adverse effects of the procedure.", "keywords": [ "browlift", "minimally invasive", "surgical technique", "frontalis suspension", "outpatient procedure", "non-excisional browlift" ], "content": "Introduction\n\nPtosis of the forehead or eyebrows is a common problem, especially among the elderly. Using the muscles that move the eyebrows, a lot of expressions can be communicated and can be easily recognized. Laterally inclining eyebrows transmit sadness, medially inclining eyebrows transmit anger, low eyebrows transmit tiredness, drawn up eyebrows transmit surprise and properly aligned eyebrows transmit an alert, rested state allowing the mouth to smile1,2. The position of the brow is affected by the corrugator supercilii, depressor supercilii, orbicularis oculi, and procerus (brow depressor muscles) and the frontalis muscle (brow elevator muscle)3. The motor innervation of these muscles is supplied by branches of the facial nerve. The temporal branch innervates the frontalis, superior part of the orbicularis oculi, the transverse head of the corrugator supercilli and superior part of the procerus muscles. The zygomatic branch innervates the inferior and medial parts of the orbicularis oculi, the inferior part of the procerus, the depressor supercilii and the oblique head of the corrugator supercilii muscles.\n\nCommon complaints of brow ptosis are a tired and heavy feeling of the eyes, problems watching television and reading, increased tearing and a limited field of vision1–6. Continuous activation of the scalp and forehead muscles may additionally cause tension headaches and eyestrain4. On closer examination, the cause of this may be partly due to a too low position of the eyebrows. As a result, the skin of the upper eyelids is pushed down, as it were, so that it looks like there is too much skin there. In patients with brow ptosis, unintended emotions can be shown, which can be misinterpreted by others5,6.\n\nIf the eyebrows are in a low position, a blepharoplasty alone makes little sense and sometimes no improvement occurs. It is better to first correct the cause of the deviation, the low position of the eyebrows, and then remove any remaining skin surplus from the upper eyelids. A wrong assumption is that the simultaneous treatment of blepharochalasis, and thus including a blepharoplasty procedure to the browlift procedure is not deemed beneficial. The added removal of upper eyelid skin in a single operation actually worsens the patient’s appearance due to excessive traction of the skin, causing an increase in their brow ptosis giving the patient a tired, older and angry appearance5,7,8. Therefore, patients desire a difference in brow stance that is both long-lasting and appears natural. Other factors that are included in decision-making are the visibility of scars, the cost of the procedure and practicality (procedure time, use of anaesthetics etc.).\n\nSeveral techniques exist to treat ptosis of the eyebrows5,9–18. Some only treat brow ptosis, and other techniques are also used for treating a wide spectrum of facial aging changes. Direct browlift and the traditional fascial suspension technique tend to leave a very noticeable scar above the eyebrow15–18. Other options such as the (mid-) forehead browlift and coronal browlift can be used to elevate the brow in patients with deep creases who are not candidates for extensive surgical procedures, although significant scars are still made19.\n\nForeheadplasty, or open browlift, techniques have also been used to lift the eyebrows, namely the forehead (pretrichial) incision, corobregmatic incision, vertex incision, lambdoidal incision, W-incisions, lambdoidal paddle incision and the interlocking Ms5. These techniques take the same time as an endoscopic procedure and have the ability to adapt to various wrinkle, crease and forehead hairline considerations5. Other techniques, such as the transblepharoplasty or transpalpebral approach, use an upper blepharoplasty to resect the corrugator muscles and divide the procerus muscle along with temporal incisions to elevate the lateral eyebrow20,21.\n\nIn 1994 Vasconez et al.22 first described the endoscopic browlift technique, which has been widely studied10–12,14,23–32. This is a popular technique since advantages include less scarring, alopecia and numbness posterior to the scar. Despite a 70% satisfaction rate reported, the frequency of the endoscopic lift being performed by plastic surgeons has decreased33. The endoscopic browlift is also a costly and lengthy procedure that requires an operation theatre and can’t be performed on an outpatient basis. Additionally, reduced sensation has been described in endoscopic and open browlifts34.\n\nAlthough many options exist in the treatment of brow ptosis, there is of yet no golden standard. The technique developed by Dr. L.T. Tan presented in our previous article35 combines traditional browlift approaches, such as the incision sites of the pretrichial and direct browlift without excising skin, with the dissection of frontalis muscle from the periost as used in the endoscopic browlift. Our technique found high satisfaction rates on scars (72.4%) and symptom improvement (72.4%) with minimal complications (long-lasting pain (n=4), cosmetic deterioration (n=3), noticeable subcutaneous lump (n=3), photophobia (n=1) and numbness (n=1)) and, in addition, does not require an operation theatre since only local anaesthesia is used. Thus our technique proves to be a more practical, cheaper alternative with optimal functional outcome and minimal scarring and therefore an optimal cosmetic result, especially when compared to more traditional browlift techniques. The aim of this study is to further evaluate the clinical and cosmetic data from patients who will undergo this procedure in the future and provide for more accurate measurement of the surgical effects.\n\nThe majority of the brown ptosis cases occur as weakening (e.g. involutional changes) or descent of the periorbital and/or facial soft tissue. This typically occurs at the temporal side of the brow first, mainly and the most temporal 1/3 of the eyebrow. Anatomically, the frontalis muscle raises the eyebrow and the frontal branch of the facial nerve is responsible for the innervation of this muscle. Normally the frontalis muscle lifts the medial 2/3 of the eyebrow and with increasing age, laxity of tissues (in particular collagen) combined with the descent of facial/periorbital soft tissue, patients develop lateral brow droop.\n\nIn terms of differential diagnosis, brow ptosis can occur with the following diseases: 1) paralysis or weakness of the frontalis muscle (facial nerve palsy, Bell’s palsy, acoustic neuroma, surgical trauma, birth trauma, congenital diseases1, myasthenia gravis, myotonic dystrophy, or oculopharyngeal muscular dystrophy); 2) involuntary contraction of the orbicularis oculi (blepharospasm or facial dystonias); and 3) mechanical causes, which can result in descent of the brow (neoplasms: basal cell carcinoma; squamous cell carcinoma; keratoacanthoma or melanoma).\n\nThere is a need to provide relevant evidence in patient care in (aesthetic) brow surgery, since most browlift techniques are done from experience and not necessarily evidence based. So therefore we took a number of key questions into account:\n\n1. Will the browlift relieve complaints (increase worthwhileness)?\n\n2. What is the longevity of the procedure?\n\n3. How natural will the forehead region appear after surgery? This will be assessed via:\n\na. Shape of the forehead\n\nb. Visibility of scars\n\nc. Abnormal wrinkle showing as a result of pulled up frontal muscles\n\n4. Will there be any unforeseen adverse effects? This will be assessed via numbness.\n\nThe primary objective of this study is to determine if our browlift technique is comparable or superior to existing types of browlifts using facial observation questionnaires administered pre- and postoperatively:\n\nFACE-Q\n\nPre-operative\n\n▪ Age appraisal VAS\n\n▪ Age Appearance Appraisal\n\n▪ Face Overall\n\n▪ Forehead and Eyebrows\n\n▪ Forehead Eyebrows Scalp\n\n▪ Lines Forehead\n\n▪ Psychological\n\nPost-operative\n\n▪ Age appraisal VAS\n\n▪ Age Appearance Appraisal\n\n▪ Decision\n\n▪ Face Overall\n\n▪ Forehead and Eyebrows\n\n▪ Forehead Eyebrows Scalp\n\n▪ Lines Forehead\n\n▪ Psychological\n\n▪ Outcome\n\nSF-36\n\n\nProtocol\n\nResearch design. A prospective observational study will be performed on all available data from patients who undergo a browlift procedure in the Haaglanden Medical Center (HMC) from 1-6-2021 until 31-5-2022. The Medical Ethics board of the Haaglanden Medical Center judged that this research (protocol number N21.009/ML/ml) is mainly an evaluation of a common treatment method and therefore does not need a formal ethics approval procedure. Since this is an exploratory pilot study, no formal power and sample size calculation was done and we aim to include at least 50 patients (1 per week)\n\nPatients with medical (brow-ptosis, facial paralysis i.e.) or cosmetic indication.\n\nSufficient understanding of the Dutch or English language\n\nWillingness to participate in extra study specific follow-up moments and filling in of questionnaires\n\nInsufficient understanding of the Dutch/English language\n\n<18 years of age\n\nPatients with previous brow or eyelid surgery\n\nHistory\n\nEvery patient that presents with droopy eyelids and/or eyebrows should undergo a thorough medical and family history, including at least the following aspects:\n\nSlowly progressive onset of the symptoms, together with a positive family history can indicate myotonic dystrophy or oculopharyngeal dystrophy.\n\nAssessment of possible fluctuation of the symptoms or presence of fatigue (which can be present in case of myasthenia gravis).\n\nA detailed history of surgery and/or trauma may point in the direction of damage to the frontal nerve or muscle scarring.\n\nSymptoms that could indicate a facial palsy.\n\nA detailed oncological history, stroke and/or head and neck tumours.\n\nPhysical examination\n\nA complete plastic ophthalmic examination should be conducted in every patient, including:\n\n- A visual acuity, pupillary and extraocular investigation\n\n- A neurological examination of each cranial nerve\n\n- The mentioned distances in figure 1 will be noted and measured\n\n- A complete skin examination including if there is skin resting in the eye lashes\n\n- The brow position will be noted in the situation of a relaxed frontalis muscle\n\n- Possible prominent dynamic and static rhytids will be checked\n\n- The location of the hairline will be noted\n\n- In case of a paralytic brow, the physician will check for signs of facial trauma and/or scarring\n\nAll patients will be evaluated to check if there is dermatochalasis/ptosis and concomitant brow ptosis. Additionally, every patient scheduled for blepharoplasty will be checked if brow-repositioning surgery is necessary.\n\nPre-operatively the patient is informed about the procedure and information is given about (visible) scar formation, asymmetry and post-operative pain.\n\nThe patient is evaluated in the upright position. The degree of ptosis and position of the hairline is noted and compared with the contralateral side. Manual elevation of the brow to the ideal position will help to determine how much of the visual field deficit is secondary to the brow ptosis alone. Since exuberant brow lifting may compromise eye closure, attention should be paid to any degree of lagophtalmos if present. Secondly, injections with xylocaine + adrenalin 1:100.000 are administered into the dermis and subdermal until the periost of the lateral frontalis muscle region for adequate local anaesthesia. Two horizontal incisions (approximately 1-1.5 cm) are made, just above the lateral 1/3 of the eyebrow and the other just cranial to the hairline in line with the frontalis muscle. These locations are chosen to avoid injury to the frontal nerve (Figure 1). Secondly, the frontalis muscle is bluntly dissected from the underlying periost. The frontalis muscle is then suspended 2–5mm and fixated at the cranial site to the periost of the frontal bone using a 3-0 (Ethilon) suture. Then the muscle is also suspended 2–5mm at the caudal site using a 3-0 (firstly Ethilon, later Prolene) suture fixing it to the periost of the frontal bone. Attention is paid to achieve perfect symmetry between both sides. Finally, the skin is closed with Ethilon 5-0 sutures. The head is than wrapped with a bandage for 24–48 hours. The duration of the procedure is 20–30 minutes. For graphical depiction of technique, see Figure 2.\n\nMeasured with Emotrics software. *Informed consent was obtained for the usage of the patient’s photograph\n\nBLCD= Brow-lateral canthal distance, BMD= Brow-lid margin distance, MPD= Lid margin-pupil distance, BPD= Brow-pupil distance, BMCD= Brow-medical canthal distance, CAD= Canthal-nasal alar distance, BAD= Brow alar distance, PF= palpebral fissure height. All distances to the pupil are measured to the centre of the pupil\n\nSurgical complications are relatively uncommon. However, bleeding, numbness and tingling, injury to the facial nerve resulting in paralytic brow ptosis, infection and postoperative asymmetry have been described in previous browlift studies (25–35) and will be documented accordingly.\n\nPatients with an indication for brow correction will receive information to review study specific information. Two weeks after patients have received the study information, they will be asked to participate in our study. see Table 1 for patient assessment moments.\n\nDemographic information of patients will also be collected, such as age, race, and gender, as well as surgical indication and level of brow depression (see Figure 1).\n\nVectra XT. The Vectra XT 3D-imaging device (M3) produced by Canfield can be used for a wide variety of medical indications where accurate measurements can provide clear and clinically relevant information for both the practitioner and the patient.\n\nA 3D-image can be easily made with the Vectra XT by correct positioning of the patient in front of the device. The device provides standard outlines, so correct positioning can be achieved relatively easily. The device and its accompanying software subsequently provide the practitioner with some standard measurements, which can be supplemented with additional measurements. (see Figure 1 for the overview). Additionally, the Vectra XT software can automatically provide before and after surgery differences in facial measurements by overlaying the produced photographs within one patient file.\n\nPhotographer instructions (Table 2)\n\n- 3D imaging photographs can only be made at HMC Bronovo, where the Vectra XT is situated.\n\n- Create a separate patient file for every patient, where the different assessment moments can be safely stored and lastly compared.\n\n- Ensure correct positioning: align the patient’s eyes within the horizontal and vertical green bars which are provided within the standard Face Sculptor software.\n\n- Inform the patient to sit still looking straight forward, ensuring that the patient looks at their own eyes in the centre of the mirror on the device.\n\n- Once the 3D picture is made, measure the parameters as depicted in Figure 1 and save the photograph within the patient file clearly stating the assessment moment.\n\nPatient instructions\n\nTo ensure high quality photos and for the best comparison it’s important that the patient has a neutral facial expression and is not wearing (or is wearing minimal) make-up.\n\n- The patient should be seated to ensure stable positioning for optimal photography.\n\n- To have a clear view of the patient’s face, their hair needs to be tied or tucked away such that none of the observed facial features are hidden.\n\n○ It’s advisable to have some disposable hair clips for patients with long hair.\n\n1. Scarring after procedure\n\n2. Functionality of eyebrow movement\n\n3. Amount of correction in brow ptosis, measured in VECTRA\n\n4. Longevity of procedure in months\n\n5. Aesthetic result as assessed by questionnaires\n\n6. Adverse effects of procedure\n\nFor analyses we will use descriptive statistics and inferential statistics. A Kolmogorov-Smirnov test, a Q-Q plot and Levene’s test will first test all data for normality. Categorical variables will be expressed as n (%). Continuous normally distributed variables will be expressed by their mean and standard deviation, not normally distributed data by their median and interquartile range for skewed distributions. To test groups, categorical variables will be tested using the Pearson’s Chi-square test or Fisher’s exact test, when appropriate. Normally distributed continuous data will be tested with the independent samples Students t-test and in case of skewed data, with the independent samples Mann-Whitney U-test. Not normally distributed data will be tested by a Log rank test. When appropriate, for testing multiple possible factors for survival, a Cox proportional hazards analysis will be used. Statistical Package for Social Sciences (SPSS, Chicago, IL, USA Version 20.0) will be used to prepare the database and for statistical analysis.\n\nThe local data originating from the HMC-population will be coded. Each subsequent included and eligible case will be given a number, which is linked to the identifying patient details. The key to translate the code will be held by the main investigator in the HMC, Dr. de Jongh. The coded data will be stored by the aforementioned local investigators of this study in a spreadsheet that is secured by a password only known by these investigators. Only they will therefore have access to this data. Data will be kept in storage for 15 years. After the study ends the data will be made available on request. Each request will be assessed by dr. de Jongh.\n\n\nConclusion\n\nThis study will evaluate the quality of life related to and quantified data for this new browlift procedure. This will hopefully lead to an increased number performed under local anesthesia in an ambulatory setting. Follow-up studies should investigate our browlift technique in specific patient subgroups (for example patients with a peripheral facial palsy).\n\n\nStudy status\n\nOngoing; completion is expected in the first quarter of 2023.\n\n\nDissemination\n\nThe results of this study will be presented at scientific meetings and published in peer-reviewed medical journals.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nfigshare: Data Protocol Browlift Study. https://doi.org/10.6084/m9.figshare.17207579.v136\n\nThis project contains the following files:\n\n- FACE-Q Age Appearance Appraisal.pdf\n\n- FACE-Q Age Appraisal VAS.pdf\n\n- FACE-Q Decision.pdf\n\n- FACE-Q Face Overall.pdf\n\n- FACE-Q Forehead and Eyebrows.pdf\n\n- FACE-Q Lines Between Eyebrows.pdf\n\n- N21.009 P1a. Verklaring niet WMO d.d. 09-04-2021[24407].pdf\n\n- Proefpersoneninformatie voor deelname Wenkbrauwlift Versie 2. 17-03-2020.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author contributions\n\n\n\nIdea for the study: FdJ, SP, KW, LT\n\nDeveloping study design with appropriate outcome measurements: FdJ, SP, LK, ES, KI, KW, LT\n\nIn hospital logistics: FdJ, LK, ES\n\nDrafting the protocol: FdJ, LK, ES, SP, KI, KW, LT\n\nFinal approval: FdJ, LK, ES, SP, KI, KW, LT\n\n\nReferences\n\nEllenbogen R: Transcoronal eyebrow lift with concomitant upper blepharoplasty. Plast Reconstr Surg. 1983; 71(4): 490–9. PubMed Abstract \| Publisher Full Text\n\nEllenbogen R: Medial brow lift. Ann Plast Surg. 1980; 5(2): 151–2. PubMed Abstract \| Publisher Full Text\n\nLee TS, Wang L, Han R, et al.: Options in Repositioning the Asymmetric Brow from Paralysis and Trauma. Facial Plast Surg. 2017; 33(6): 627–38. PubMed Abstract \| Publisher Full Text\n\nFinsterer J: Ptosis: causes, presentation, and management. Aesthetic Plast Surg. 2003; 27(3): 193–204. PubMed Abstract \| Publisher Full Text\n\nSundine MJ, Connell BF: The Open Browlift. Facial Plast Surg. 2018; 34(2): 128–38. PubMed Abstract \| Publisher Full Text\n\nConnell BF, Marten TJ: The male foreheadplasty. Recognizing and treating aging in the upper face. Clin Plast Surg. 1991; 18(4): 653–87. PubMed Abstract\n\nFlowers RS, Ceydeli A: The open coronal approach to forehead rejuvenation. Clin Plast Surg. 2008; 35(3): 331–51; discussion 29. PubMed Abstract \| Publisher Full Text\n\nMatarasso A: Endoscopically assisted forehead-brow rhytidoplasty: theory and practice. Aesthetic Plast Surg. 1995; 19(2): 141–7. PubMed Abstract \| Publisher Full Text\n\nTroilius C: A comparison between subgaleal and subperiosteal brow lifts. Plast Reconstr Surg. 1999; 104(4): 1079–90; discussion 91-2. PubMed Abstract\n\nDayan SH, Perkins SW, Vartanian AJ, et al.: The forehead lift: endoscopic versus coronal approaches. Aesthetic Plast Surg. 2001; 25(1): 35–9. PubMed Abstract \| Publisher Full Text\n\nMcKinney P, Sweis I: An accurate technique for fixation in endoscopic brow lift: a 5-year follow-up. Plast Reconstr Surg. 2001; 108(6): 1808–10; discussion 11-4. PubMed Abstract \| Publisher Full Text\n\nJones BM, Grover R: Endoscopic brow lift: a personal review of 538 patients and comparison of fixation techniques. Plast Reconstr Surg. 2004; 113(4): 1242–50; discussion 51-2. PubMed Abstract \| Publisher Full Text\n\nSidle DM, Loos BM, Ramirez AL, et al.: Use of BioGlue surgical adhesive for brow fixation in endoscopic browplasty. Arch Facial Plast Surg. 2005; 7(6): 393–7. PubMed Abstract \| Publisher Full Text\n\nPapadopulos NA, Eder M, Weigand C, et al.: A review of 13 years of experience with endoscopic forehead-lift. Arch Facial Plast Surg. 2012; 14(5): 336–41. PubMed Abstract \| Publisher Full Text\n\nRafaty FM, Goode RL, Fee WE Jr: The brow-lift operation. Arch Otolaryngol. 1975; 101(8): 467–8. PubMed Abstract \| Publisher Full Text\n\nJohnson CM, Jr., Anderson JR, Katz RB: The brow-lift 1978. Arch Otolaryngol. 1979; 105(3): 124–6. PubMed Abstract \| Publisher Full Text\n\nLewis JR Jr: A method of direct eyebrow lift. Ann Plast Surg. 1983; 10(2): 115–9. PubMed Abstract \| Publisher Full Text\n\nUeda K, Harii K, Yamada A: Long-term follow-up study of browlift for treatment of facial paralysis. Ann Plast Surg. 1994; 32(2): 166–70. PubMed Abstract \| Publisher Full Text\n\nAngelos PC, Stallworth CL, Wang TD: Forehead lifting: state of the art. Facial Plast Surg. 2011; 27(1): 50–7. PubMed Abstract \| Publisher Full Text\n\nKnize DM: Limited-incision forehead lift for eyebrow elevation to enhance upper blepharoplasty. Plast Reconstr Surg. 1996; 97(7): 1334–42. PubMed Abstract \| Publisher Full Text\n\nKnize DM: Limited incision forehead lift for eyebrow elevation to enhance upper blepharoplasty. Plast Reconstr Surg. 2001; 108(2): 564–7. PubMed Abstract \| Publisher Full Text\n\nVasconez LO, Core GB, Gamboa-Bobadilla M, et al.: Endoscopic techniques in coronal brow lifting. Plast Reconstr Surg. 1994; 94(6): 788–93. PubMed Abstract \| Publisher Full Text\n\nde la Fuente A, Santamaría AB: Endoscopic forehead lift: is it effective? Aesthet Surg J. 2002; 22(2): 113–20. PubMed Abstract \| Publisher Full Text\n\nIblher N, Manegold S, Porzelius C, et al.: Morphometric long-term evaluation and comparison of brow position and shape after endoscopic forehead lift and transpalpebral browpexy. Plast Reconstr Surg. 2012; 130(6): 830e–40e. PubMed Abstract \| Publisher Full Text\n\nGraf RM, Tolazzi AR, Mansur AE, et al.: Endoscopic periosteal brow lift: evaluation and follow-up of eyebrow height. Plast Reconstr Surg. 2008; 121(2): 609–16; discussion 17–9. PubMed Abstract \| Publisher Full Text\n\nGraf R: Discussion: Morphometric long-term evaluation and comparison of brow position and shape after endoscopic forehead lift and transpalpebral browpexy. Plast Reconstr Surg. 2012; 130(6): 841e–2e. PubMed Abstract \| Publisher Full Text\n\nJavidnia H, Sykes J: Endoscopic brow lifts: have they replaced coronal lifts? Facial Plast Surg Clin North Am. 2013; 21(2): 191–9. PubMed Abstract \| Publisher Full Text\n\nJones BM, Lo SJ: The impact of endoscopic brow lift on eyebrow morphology, aesthetics, and longevity: objective and subjective measurements over a 5-year period. Plast Reconstr Surg. 2013; 132(2): 226e–38e. PubMed Abstract \| Publisher Full Text\n\nBehmand RA, Guyuron B: Endoscopic forehead rejuvenation: II. Long-term results. Plast Reconstr Surg. 2006; 117(4): 1137–43; discussion 44. PubMed Abstract \| Publisher Full Text\n\nGuyuron B: Endoscopic forehead rejuvenation: I. Limitations, flaws, and rewards. Plast Reconstr Surg. 2006; 117(4): 1121–33; discussion 34–6. PubMed Abstract \| Publisher Full Text\n\nRamirez OM: Why I prefer the endoscopic forehead lift. Plast Reconstr Surg. 1997; 100(4): 1033–9; discussion 43–6. PubMed Abstract \| Publisher Full Text\n\nStanek JJ, Berry MG: Endoscopic-assisted brow lift: revisions and complications in 810 consecutive cases. J Plast Reconstr Aesthet Surg. 2014; 67(7): 998–1000. PubMed Abstract \| Publisher Full Text\n\nChiu ES, Baker DC: Endoscopic brow lift: a retrospective review of 628 consecutive cases over 5 years. Plast Reconstr Surg. 2003; 112(2): 628–33; discussion 34–5. PubMed Abstract \| Publisher Full Text\n\nGuillot JM, Rousso DE, Replogle W: Forehead and scalp sensation after brow-lift: a comparison between open and endoscopic techniques. Arch Facial Plast Surg. 2011; 13(2): 109–16. PubMed Abstract \| Publisher Full Text\n\nDe Jongh FW, Sanches EE, Kooiman L, et al.: A new minimally invasive surgical browlift technique with minimal scarring. Journal of plastic, reconstructive & aesthetic surgery: JPRAS. 2021; Submitted.\n\nPouwels S: Data Protocol Browlift Study. figshare. Journal contribution. 2021. http://www.doi.org/10.6084/m9.figshare.17207579.v1" }	[ { "id": "142355", "date": "01 Jul 2022", "name": "Yinon Shapira", "expertise": [ "Reviewer Expertise Oculoplastic surgery", "Orbital Disease and surgery", "Lacrimal Disease and surgery", "Bio-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 authors present a protocol of a study aimed to prospectively evaluate a new \"minimal invasive\" browlift technique. The motivation for the described prospective study (in this protocol) stems from a previous study conducted by the authors (which I assume was retrospective?) showing favorable results. The previous paper (De Jongh FW, Sanches EE, Kooiman L, et al.: A new minimally invasive surgical browlift technique with minimal scarring. Journal of plastic, reconstructive & aesthetic surgery: JPRAS. 2021; Submitted), does not seem to have been published yet, so is unavailable for scrutiny. The major strengths of this study protocol include the use of reproducible photographic assessment/measurements for outcomes, and also the very long follow-up (5 years) for assessment of longevity of the surgical correction. Also, the use of subjective questionnaires makes the outcome assessment (objective and subjective) overall robust. Nonetheless, given a previous paper by the same group describing this technique and showing preliminary results, I am not sure that publishing a protocol of a currently running prospective study is of significant interest. It would make more sense to publish a review article with an overview of brow lift techniques (their mechanisms, strengths, and limitations), therein introducing the new technique, and briefly describing the future directions (e.g. the prospective study underway). Furthermore, the authors state that “this is an exploratory pilot study” – given the results of the first study that has been submitted, and given the conclusions stated by the authors: “our technique proves to be a more practical, cheaper alternative with optimal functional outcome and minimal scarring and therefore an optimal cosmetic result, especially when compared to more traditional browlift techniques”, ideally a randomized trial to compare the outcomes of the novel technique to more traditional approaches should have been planned. If randomization is impractical, at least a prospective comparison between techniques is merited.\nRegarding the investigated novel brow-lift technique, the description of surgical technique is not completely clear. The figure only depicts the incision lines. My main concerns:\nThe “lateral” suprabrow incision, as seen in the figure, is not significantly smaller than the approach many surgeons now advocate when performing a direct brow lift. As we try to avoid the supraorbital nerve, combined with the fact that brow-ptosis is usually less prominent in the medial brow (thus lift and contouring are focused on the more lateral aspects), direct brow-lifts incisions are often performed mid-laterally. Therefore, the presented technique does not seem to spare the patients a considerable length of supra-brow incision.\n\nIn this technique \"the frontalis muscle is bluntly dissected from the underlying periost. The frontalis muscle is then suspended 2–5mm and fixated at the cranial site to the periost of the frontal bone.\" In order to achieve a meaningful brow-lift, the pretrichial and endoscopic brow-lift approaches necessitate dissection all the way down (and over) the superior orbital rim. Even then (and in the sub-periosteal plain), the amount of lift and longevity of lift are somewhat inferior to the direct lift approach (due to the mechanical advantage as the incisions are close to the brows). Furthermore, the authors’ technique seems to involve dissection in the sub-aponeurotic plane, which would mean that for an adequate lift the periosteum would need to be elevated at the region of the superior orbital rim (and sub-brow). There is no mention of these considerations in the technique description. It might be described in more details in the paper describing the previous results (but is yet to be published).\n\nFurther specific points:\nAbstract: \"The aim of this study is to prospectively evaluate the new minimal invasive (MINE) browlift technique\" - Should it rather be \"a new\"?\n\nSince the current focus of this paper/protocol is not to be a general review, the introduction is long and contains many general details (e.g. details of anatomy and a passage describing why blepharoplasty should not be performed for treating brow ptosis) that do not serve the scope of the paper – It should be much more focused and concise dealing only with the aspects of the technique described. In fact, the last paragraph of the introduction almost suffices in “introducing” the scope of the paper.\n\nTypo: “The majority of the brown ptosis cases…”.\n\n“Will there be any unforeseen adverse effects? This will be assessed via numbness.” Should add assessment of injury to the frontal branch of the facial nerve.\n\nPhysical examination: Will any of the brow-lifts be combined with upper blepharoplasty? If so, Bells phenomenon, lagophthalmos and assessment of the lids should be done.\n\nPhotographer instructions and Patient instructions: These sections add little to the understanding of the project, and are somewhat overly technical (seems more fitting for the in-house protocol for the study execution). Table 2 should suffice (perhaps as a supplement) in the context of publication.\n\nData Analysis: \"Normally distributed continuous data will be tested with the independent samples…\" The assessment should rather be for dependent samples as this would be a within-subject design (before-after) and not a comparison between case-control.\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? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/11-207
https://f1000research.com/articles/10-521/v1	30 Jun 21	{ "type": "Method Article", "title": "Numerical model for enhancing stimulated Brillouin scattering in optical microfibers", "authors": [ "Soon Heng Yeap", "Siamak Dawazdah Emami", "Hairul Azhar Abdul-Rashid", "Soon Heng Yeap", "Siamak Dawazdah Emami" ], "abstract": "Stimulated Brillouin scattering (SBS) is useful, among others for generating slow light, sensing and amplification. SBS was previously viewed as a poor method due to the limitation on optical power in high-powered photonic applications. However, considering the many possible applications using SBS, it is now of interest to enhance SBS in areas of Brillouin frequency shift together with Brillouin Gain. A numerical model, using a fully vectorial approach, by employing the finite element method, was developed to investigate methods for enhancing SBS in optical fiber. This paper describes the method related to the numerical model and discusses the analysis between the interactions of horizontal, shear and hybrid acoustic modes; and optical modes in optical fiber. Two case studies were used to demonstrate this. Based on this numerical model, we report the influence of core radius, clad radius and effective refractive index on the Brillouin frequency shift and gain. We observe the difference of Brillouin shift frequency between a normal silica optical fiber and that of a tapered fiber where nonlinearities are higher. Also observed, the different core radii used and their respective Brillouin shift. For future work, the COMSOL model can also be used for the following areas of research, including simulating “surface Brillouin shift” and also to provide in-sights to the Brillouin shift frequency vB of various structures of waveguides, e.g circular, and triangular, and also to examine specialty fibers, e.g. Thulium and Chalcogenide doped fibers, and their effects on Brillouin shift frequency.", "keywords": [ "Stimulated Brillouin Scattering", "Brillouin Shift Frequency", "Effective Refractive Index", "COMSOL", "Slow Light" ], "content": "Introduction\n\nStimulated Brillouin scattering (SBS) is a nonlinear process caused by acoustic phonon scattering propagating in the backward direction. Acoustic vibration across mediums scatters the incident light of the pump wave causing an acoustic frequency shift resulting in Stokes and anti-Stokes waves. The process of transferring energy from the pump wave to the Stokes wave is known as the scattering phenomenon. The Stokes wave counter propagates in the opposite direction to the pump wave. The presence of acoustic waves propagating on the medium’s surface is known as the surface acoustic wave. SBS theory was first explained by Leon Brillouin in 1922, and since then related experimental works have been performed in the past decade.1 This paper describes a numerical model that analyzes the interactions of horizontal, shear and hybrid acoustic modes; and optical modes in optical fibers. The numerical model was developed using COMSOL Multiphysics. Two case studies were used to demonstrate the model’s utility. Based on this numerical model, we report the influence of core radius, clad radius and effective refractive index on the Brillouin frequency shift and gain coefficient.\n\n\nLiterature review\n\nPrior studies related to SBS have focused on the acoustic frequency shift of different types of fibers. In theory, peak Brillouin gain for a standard silica fiber is approximated to be 5 × 10−11 m/W.2 In addition, fibers around 2.6 × 10−12 m/W have been presented by Nikles et al.3 Optimized SBS acoustic frequency shift for tellurite photonic crystal fiber (PCF) was recorded at 8.43 GHz, which gives 9.48 × 10−11 m/W of Brillouin gain.4 Experimental results for SBS across a chalcogenide fiber was demonstrated by Song et al. at 6.08 × 10−9 m/W, showing a higher Brillouin gain compared to a standard silica fiber.5 Woodward et al. reported an experimental study on SBS in small-core PCF,6 which discussed the complexity of acoustic wave dynamics for different wavelengths of light, overlap between optical waves were minimum at 532 nm. Consequently, at 1550 nm, a higher overlap is achieved, which contributes to a higher Brillouin gain, and therefore a lower threshold power of 1160 mW.6 More recently, Tchahame et al. demonstrated a multimode Brillouin spectrum across a long tapered PCF.7 Beugnot et al. successfully conducted both the first numerical and experimental work on surface acoustic waves (SAW) of silica microfiber in 2014, with a Brillouin gain equivalent to 1.4 × 10−12 m/W.8 The idea is greatly appreciated as SAW has outstanding potential contribution in the optical sensor due to its sensitivity on various physical perturbations. However, the limitation is that threshold power of the tapered fiber is relatively high and so it is not feasible in optical application. Acoustic confinement in the fiber core is required to ensure high overlap with the optic wave. Cherif et al. studied the Brillouin spectrum of SBS characterization for small core tellurite PCF with variation in air-filling ratio.4 In addition, previous results by Hu et al. showed a low threshold power of 52 mW on chalcogenide fibers whose acoustic waves were confined to the core.9 However, for SBS across tapered silica fibers an undesired high threshold power was observed due to a reduced interaction of the surface acoustic wave with the optical wave. To counter this effect, gold and silver cladding materials have been proposed. The finding from Kim et al. shows novel shear Brillouin scattering detection using microscopic resolution.10 This finding is fundamental as previous experimental set ups focused on longitudinal acoustic wave propagation.\n\nSeveral experimental studies have been carried out by researchers to study the behavior of SBS in optical fibers. However, there are several limitations in experimental studies such as fabrication certainty, environmental influences and access to laboratory equipment. An accurate modeling tool, developed for the purpose of aiding experimental studies, would be beneficial to speed up research while preserving a good level of accuracy and confidence. Finite Element Method (FEM), a method that applies meshing technique to solve partial differential equations (PDE) with a certain boundary condition.14 FEM received great attention when improvements in handling boundary conditions with the implication of penalty functions was discussed.15 This gave FEM the preference in optical fiber modeling considering the boundary issue as the problem that other methods fail in. Ham et al.16 then introduced the complete numerical solution, where FEM is used with spectral method that provides accuracy and consistency for 2-D and 3-D cases involving harmonic functions. These findings made FEM more suitable for optical fiber numerical modeling. Sriratanavaree et al.17 used FEM in the study of optical and acoustic wave interaction in silicon slot waveguides. Subsequently, Monfared et al.18 showed how FEM modeled for composite behavior of bond particle at fiber interface. Findings from Liu et al.19 showed a numerical solution using FEM to enumerate the tension and bending in optical fiber accurately. A report on optical fiber modeling and simulation of effective refractive index for tapered fiber with finite element method was deliberated by Lee et al.20 Rahman et al.11, 12 reported numerical modeling of SBS, considering the optical fundamental mode in the optical fiber using FEM.\n\n\nMethods\n\nWe present a numerical model to optimize Brillouin frequency shift and gain based on various core diameters of the tapered region of silica microfiber structures. To further understand the interactions, the COMSOL model should be capable of modelling solutions in the given structures of the optical fiber. Previous research was mainly done to enhance performance on spatial resolution and also sensing range, but there have not been many insights for gain. The ability to increase the Brillouin gain coefficient has opened new opportunities to control their interaction, and several new industrial and commercial applications. In this research work, we demonstrate the numerical model for a microfiber design that is expected to increase Brillouin gain coefficient. The fully vectorial method, developed in COMSOL, is used in this case to determine the contributions of various optical fibre parameters towards SBS, thus aiding the design of microfibers with enhanced SBS performance. The equation below is used for the analysis of light guidance where H is the full vectorial magnetic field, * represents the complex conjugate and transpose, ω2 denotes the eigenvalue where ω is the optical angular frequency of the wave and ε and μ are the permittivity and permeability, respectively.21\n\nEquation (1) solves for the propagation constant of optical modes in optical waveguides, which can also guide acoustic mode. The propagation constant of the optical wave, β, is defined as βoptic = 2πneff/λ. There are two basic types of acoustic waves, namely shear and longitudinal acoustic waves. Shear waves are associated with dominant material dispersion in the transverse directions, which is perpendicular to the direction of propagation, taken here as the z-axis. On the other hand, for a longitudinal wave, expansion and contraction of the wave is associated with particle movements along the z direction which is in parallel to the wave propagation. However, acoustic wave propagating through a waveguide can be a combination of shear and longitudinal acoustic waves. Brillouin frequency shift of the Stokes wave is given as f = 2neff Vac/λ, where, Vac is the acoustic velocity. The acoustic wave satisfies Hooke’s Law12 which relates to the stress (tensor) and strain (force) of the waveguiding materials. Electric field associated with a high power optical signal causes molecular movements due to electrostriction process.13 Such a material movement can generate acoustic waves that leads to density variation along the waveguide. The time and space dependent density variation changes the refractive index profile and produces a moving optical grating. This grating can reflect incoming light when its wavelength matches the spatial period of the gratings generated by the acoustic wave. Above a threshold power, if phase matching conditions are satisfied, it can inhibit forward guidance of the incoming light. The backward scattered reflected wave is frequency shifted, which explains the occurrence of the Stokes Wave. The relationship between optic and acoustic propagation for phase matching condition can be given as: Kacoustic = 2βoptic where Kacoustic is the acoustic propagation constant and this will be double of the βoptic, the optic propagation constant.\n\nFor the SBS characterization in the optical fiber, both its guided optical and acoustic modes can be obtained using the FEM. The n eff for the optical mode in a fibre for a given radius is first calculated using H-field based FEM model. Eigenvector and eigenvalue of acoustic waves are also obtained and then the acoustic mode patterns are generated. At phase matched conditions, the acoustic wave propagation constant is double the value of the optical wave propagation constant: Kacoustic = 2β optic.\n\nIn this research work, the fully vectorial approach was used to solve the optical wave equations for neff using the commercial COMSOL software. The optical parameters E(x,y) and neff, are obtained by solving the equation below:\n\nWhere ∆t is transverse Laplacian operator in the (x,y) direction, while neff is the effective refractive index of fundamental optical mode,9 directly related to Brillouin frequency shift via the Bragg condition.10 The acoustic wave, which consists of the stress and strain components, are governed by Hooke’s Law12 whereby solving the equation below would yield its displacement.\n\nwhere T denotes the stress field and S represents the force field which is equivalent to partial differentiation of displacement. cijkl is the tensor relation of elastic stiffness where i, j, kl are equivalent to propagation in x, y and z direction respectively.22 For an isotropic medium with uniform wave propagation, the elastic stiffness constant is given by the longitudinal and shear velocity that is dependent on the material properties of the optical fiber core and cladding.12\n\nFor the purpose of calculating the Brillouin gain, the overlap factor from the fully vectorial approach was used. The overlap between optical wave and acoustic wave is given in equation (4) below.21\n\nwhere Hi (x, y) is the fundamental mode in optical wave and Uj (x, y) is the displacement vector of acoustic wave. Optic-acoustic wave overlap factor is influenced by the acoustic wave’s strain field and refractive index of the optical fiber.11 Both the optical and acoustic wave vectors have to be normalized to calculate the overlap factor.\n\nThe Brillouin gain coefficient is represented by the equation below:23\n\nWhere ρ0 is fiber core density, p12 is elasto-optic coefficient which contributes to the periodic light scattering and is FWHM of acoustic wave in SBS.23\n\nMethod in COMSOL\n\nBased on the parameters in Table 1, SBS characterization using the fully vectorial approach was performed on various core diameters of the tapered region of the silica microfiber and was verified against earlier results by H. J. Lee.23 The neff were calculated for all core diameters. Refractive index for the core and clad used were 1.4502 and 1.445 respectively considering the measured values in a typical single mode fiber.21 Following that, three cases of acoustic wave, shear, longitudinal and hybrid behavior were analyzed.\n\nOptical mode solver\n\nTo obtain the neff results for the two case studies covered in this research, the optical mode equation was solved using the COMSOL RF module. In the RF module the electromagnetic waves, frequency domain physics engine was used. The geometric structures of the fibers were generated using the two dimensional space dimension. From there, the parameters as denoted in Table 1 were entered into the solver. The density, refractive index of both the core and clad values are declared under the materials section. Thereafter, the mode analysis frequency is set to the desired wavelength of 1550 nm and the perfect electrical conductor boundary condition was used. As with all FEM solvers meshing is required. For this case, the triangular mesh and the “finer” element size was used. The effective mode index or neff for the fundamental mode can thereafter be obtained after computing the solver.\n\nAcoustic mode solver\n\nAs for the acoustic model, the acoustic waves of shear and longitudinal were evaluated independently to record the findings. Thereafter, the hybrid acoustic wave across the fiber in which both shear and longitudinal acoustic waves co-exist were examined. The hybrid mode model was the model used to determine the Brillouin Shift Frequency vB as both these waves propagate together in real life conditions. Table 2 shows the velocity assigned for core clad region in each respective case. In the case of pure shear acoustic, longitudinal velocity for the core region were made equivalent to 5736 m/s, this is to prevent interruption of longitudinal acoustic wave. Similarly, for pure longitudinal acoustic, the shear acoustic of the core was made to 3625 m/s. For the hybrid acoustic wave, the velocities of both the longitudinal and shear waves of the clad are defined to be slightly higher than the core.21 This is to prevent interruption of acoustic wave between the two regions. The core-clad ratio was taken to be 1:3 so that the clad region is long enough to prevent wave reflection from the outer side of clad back to the inner core. Equation 3 being a partial differential equation is solved using the Mathematics physics engine. The weak form PDE interface was used to solve equation 3. Like the optical model, the geometric structures were setup and defined in the model and the values for the acoustic wave for both shear and longitudinal velocities as tabulated in Table 2 were used. The Drichelet boundary condition was used for acoustic model simulation and the mesh setting for the acoustic model is similar with the optical model whereby the triangular mesh was used with the “finer” element size used. Computing the solver would return multiple results of eigenvalues. The eigenvalue returning the fundamental mode plots would be the one selected as the Brillouin Shift Frequency vB result.\n\n\nResults and discussion\n\nThe plots in Figure 1 show the Ex, Ey and the Ez components for the optical modes. Based on the numerical model, the neff was found to be 1.431599.\n\nThe plots in Figure 2 show the Ex, Ey and the Ez components for the optical modes respectively. Based on the numerical model, the neff was found to be 1.441802\n\nFigure 3 shows the pure shear acoustic mode along the silica microfiber where it dominates along x direction. For shear acoustic mode propagation, frequency shift is at 6.61 GHz with acoustic velocity of 3578 m/s.\n\nFigure 4 shows the pure shear acoustic mode along the silica microfiber where it dominates along x direction. For shear acoustic mode propagation, frequency shift is at 6.637 GHz with acoustic velocity of 3567 m/s.\n\nFigure 5 shows the pure longitudinal acoustic mode along the silica microfiber where it dominates along z direction. For longitudinal acoustic mode propagation, frequency shift is at 6.634 GHz with acoustic velocity of 3591 m/s.\n\nFigure 6 shows the pure longitudinal acoustic mode along the silica microfiber where it dominates along z direction. For longitudinal acoustic mode propagation, frequency shift is at 6.639 GHz with acoustic velocity of 3568 m/s.\n\nFigure 7 shows the hybrid acoustic mode along the silica microfiber where it dominates along z direction. For hybrid acoustic mode propagation, frequency shift is at 6.611 GHz with acoustic velocity of 3578 m/s.\n\nFigure 8 shows the hybrid acoustic mode along the silica microfiber where it dominates along z direction. For hybrid acoustic mode propagation, frequency shift is at 6.638 GHz with acoustic velocity of 3568 m/s.\n\nFrom the two case studies simulated using the COMSOL numerical model, we find that the values of the effective refractive index generated by the optical mode model increases as the core and clad diameter increases The results are recorded in Table 3 below.\n\nFigure 9 demonstrates the relationship between neff and silica microfiber diameter. neff in this case here can be seen increasing as the core diameter of optical fiber increases. The neff value increases ranging from 1.390115 to 1.446122 for 1 μm to 6 μm core diameter of the optical fiber. The values are generated based on the optical model developed in COMSOL. The effective mode index neff is based on the fundamental mode for these fibers.\n\nFrom the results obtained and shown in Table 4 based on the acoustic model simulations, the smaller core and clad radius of an optical fiber will produce a lower Brillouin shift frequency compared to an optical fiber with a larger core and clad radii. As for the acoustic velocities observed in the optical fibers, a smaller core and clad radii produces a higher acoustic velocity. The Brillouin frequency shift is due to the optical-acoustic interaction. The overlap integral as stated in equation 4 demonstrates the overlap ratio between the acoustic and optical mode in the optical fiber. Shown below are the overlap ratios with respect to their core diameter. The overlap ratios are obtained by solving the overlap integral equation and are tabulated in Table 5.\n\nFrom the results in Table 5, it can be seen that the overlap ratio decreases as the core diameter increases.\n\nThe Brillouin gain coefficient values can be calculated by substituting the values obtained from the numerical model into the equation as denoted by Equation 5. Table 6 shows the Brillouin gain coefficient for the 2 optical fibers that were modeled.\n\n\nConclusion\n\nThe main aim of this research is to investigate the Brillouin shift in a tapered silica fiber of different core and clad radii by stimulated Brillouin scattering. To do that, numerical model simulations were developed to study the behavior of the optical wave and acoustic wave propagation in the tapered fiber region. The study on the acoustic wave, which was further divided into three other waves, namely the shear, longitudinal and hybrid mode were evaluated. The hybrid mode would produce the Brillouin shift vB which this research is interested in. As the SBS phenomenon benefits from the nonlinearities of a fiber, the present research documents the difference of Brillouin shift frequency between two different core and clad diameters of the tapered fiber region. The Brillouin Frequency Shift vB occurs at lower frequencies for a tapered fiber with smaller core and cladding dimensions. Nonlinear effects were also observed to be higher in a smaller diameter fiber based on the overlap factor and Brillouin gain reported. For future work, this COMSOL model can also be used for the following areas of research which includes simulating “Surface Brillouin Shift” and also to provide in-sights to the Brillouin shift frequency vB of various structures of waveguides, e.g. circular and triangular, and also to examine specialty fibers, e.g. Thulium and Chalcogenide, and their effects on the Brillouin shift frequency.\n\n\nData availability\n\nDRYAD: Dataset of Numerical Model For Enhancing Stimulated Brillouin Scattering In Optical Fibers, https://doi.org/10.5061/dryad.kd51c5b4w.24\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nThe COMSOL software is proprietary and requires a subscription for use. The work presented in this article could instead be replicated using the open software tool FreeFEM (https://freefem.org/).", "appendix": "References\n\nL B: Diffusion de la lumière et des rayons x par un corps transparent homogène. influence de l’agitation thermique, Annual Physic. 1922; 17: 88–122. Publisher Full Text\n\nVysloukh V: Nonlinear fiber optics.1990; Vol. 160.\n\nNikles M, Thévenaz L, Robert PA: Simple distributed fiber sensor based on brillouin gain spectrum analysis. Optics Letters . 1996; 21(10): 758–760. Publisher Full Text\n\nCherif R, Salem AB, Saini TS, et al.: Design of small core tellurite photonic crystal fiber for slow-light-based application using stimulated brillouin scattering. Optical Engineering. 2015; 54(7): 75101–75101. Publisher Full Text\n\nSong KY, Abedin KS, Hotate K, et al.: Highly efficient brillouin slow and fast light using as2se3 chalcogenide fiber. Opt Express . 2006; 14(13): 5860–5865. PubMed Abstract \| Publisher Full Text\n\nWoodward RI, Kelleher EJR, Popov SV, et al.: 2014.\n\nTchahame J, Beugnot J, Maillotte H, et al.: Multimode brillouin scattering in a long tapered photonic crystal fiber, The European Conference on Lasers and Electro-Optics.2015; 25.\n\nBeugnot JC, Lebrun S, Pauliat G, et al.: 2014.\n\nHu K, Kabakova IV, Büttner T, et al.: Low-threshold brillouin laser at 2 mm based on suspended-core chalcogenide fiber. Opt Lett . 2014; 39(16): 4651–4654. PubMed Abstract \| Publisher Full Text\n\nKim M, Besner S, Ramier A, et al.: Shear Brillouin light scattering microscope.2016; 24(1): 319–328. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman BMA, Agrawal A: Finite Element Modeling Methods for Photonics. Artech House. 2013.\n\nRahman BMA, Davies JB: Finite-element solution of integrated optical waveguides. J Lightwave Technol . 1984; 2(5): 682–688. Publisher Full Text\n\nPoulton CG, Pant R, Eggleton BJ: Acoustic confinement and stimulated brillouin scattering in integrated optical waveguides. JOSA B . 2013; 30(10): 2657–2664. Publisher Full Text\n\nHughes TJ: The finite element method: linear static and dynamic finite element analysis.Courier Corporation; 2012.\n\nRahman BA, Davies JB: Penalty function improvement of waveguide solution by finite elements Microwave Theory and Techniques. IEEE Transactions. 1984; vol. 32, no. 8, pp. 922–928. Publisher Full Text\n\nHam S, Bathe K-J: A finite element method enriched for wave propagation problems. Computers & Structures . 2012; 94: 1–12. Publisher Full Text\n\nSriratanavaree S, Rahman B, Leung D, et al.: Rigorous characterization of acoustic-optical interactions in silicon slot waveguides by full-vectorial finite element method. Opt Express. 2014; 22(8): 9528–9537. PubMed Abstract \| Publisher Full Text\n\nMonfared V, Mondali M: Semi-analytically presenting the creep strain rate and quasi shear-lag model as well as finite element method prediction of creep debonding in short fiber composites. Materials & Design . 2014; 54: 368–374. Publisher Full Text\n\nLiu X, Liu N, Su X, et al.: Numerical analysis of fibers tensions in the siro-spinning triangle using finite element method. Fibers Polymers . 2015; 16(1): 209–215. Publisher Full Text\n\nLee HJ, Abdullah F, Emami SD, et al.: Fiber modeling and simulation of effective refractive index for tapered fiber with finite element method. 2016 IEEE 6th International Conference on Photonics (ICP), Kuching, Malaysia. 2016; pp. 1–3. Publisher Full Text\n\nSriratanavaree S: The characterisation of acoustic waves in optical waveguides.2014.\n\nGan WS: New Acoustics Based on Metamaterials.2017.\n\nLee HJ: Modelling of Stimulated Brillouin Scattering in Graphene-clad tapered fiber using Finite Element Method.\n\nAbdul-Rashid: Dataset of Numerical Model For Enhancing Stimulated Brillouin Scattering In Optical Fibers. DRYAD [dataset]. 2021. Publisher Full Text" }	[ { "id": "88747", "date": "23 Aug 2021", "name": "Gand Ding Peng", "expertise": [ "Reviewer Expertise Photonics and Optical Fibres" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEnhancing stimulated Brillouin scattering in optical fibres is of great importance in many applications such as optical fibre sensing and optical signal amplification. In this paper, the authors presented how to numerically model SBS in silica optical fibre in terms of Brillouin frequency shift and Brillouin gain coefficient - which are the two most fundamental yet important parameters of concern.\nI read through with great interest and was helped by the fact that the paper has presented a comprehensive overview of reported work from related reference papers.\nThe paper reported interesting and useful results on how fibre parameters and effective refractive index affect Brillouin frequency shift and Brillouin gain coefficient. There are issues that may need to be addressed and clarified:\nFor the two cases studied, the resulted effective indexes are found to be 1.431599 and 1.441802, respectively. I note that the core and cladding refractive Indexes used in the simulation are 1.4502 and 1.445, respectively. Normally we would expect that the effective index of optical mode to be within core and cladding indexes, i.e. between 1.4502 and 1.445. Why both the effective indexes are lower than 1.445 here?\nIn describing the method to obtain Brillouin frequency shift and gain coefficient, it is not clear how the optical - acoustic wave overlap factor (equation 4) is related to the Brillouin gain coefficient (equation 5). A simple explanation would be helpful. In addition, It would be helpful to explain how the Brillouin frequency shift is determined.\nIt is not very clear how the values of sheer and longitudinal velocities are assigned for core and clad for different cases. For example, it is stated that ‘in the case of pure shear acoustic, longitudinal velocity for the core region were made equivalent to 5736 m/s, this is to prevent interruption of longitudinal acoustic wave’. Please explain what ‘interruption’ means here. (Please note: the actual value for longitudinal acoustic wave velocity in the shear acoustic case in Table 2 is 5933, not 5736 as stated.)\nIn the abstract, it is stated that ‘This paper describes the method related to the numerical model and discusses the analysis between the interactions of horizontal, shear and hybrid acoustic modes; and optical modes in optical fiber’. But the rest of the paper ‘horizontal’ is replaced with ‘longitudinal’. It would be better to keep consistency. In addition, more analysis/discussion of the interaction of these acoustic modes would be helpful.\nIn this paper, it frequently referred to the two cases of different core sizes (with the same core/cladding ratio) as tapered fibre. This is not really tapered fibre cases anyway. They are just two straight optical fibre cases. The use of tapered fibre is a bit confusing. For example, in the conclusion, it is stated that ‘The Brillouin Frequency Shift vB occurs at lower frequencies for a tapered fiber with smaller core and cladding dimensions’. Here ‘for a tapered fiber’ seems inaccurate since the results are based on numerical simulation of a uniform fibre case.\nIn the paper, it has used ‘Brillouin gain coefficient’ and ‘Brillouin gain’ alternatively. It must be noted that they are two distinctive parameters with different units normally. For example, in the abstract, it is mentioned as ‘gain’ in the statement ‘Based on this numerical model, we report the influence of core radius, clad radius, and effective refractive index on the Brillouin frequency shift and gain’. Similarly, Table 6 should be ‘Brillouin gain coefficient’ and ‘Brillouin gain’. Perhaps ‘gain coefficient’, instead of 'Brillouin gain', is a slightly better alternative for ‘Brillouin gain coefficient’ if needed.\n\nSome of the references didn’t include the sources. For example, refs 21-23.\nFinally, the authors may check spelling and wording for better clarity For example, it is stated in the abstract that ‘SBS was previously viewed as a poor method due to the limitation on optical power in high-powered photonic applications’. It seems to me that ‘method’ is not an appropriate term here.\nIn summary, although there are aspects that the paper could be improved upon as mentioned above, the paper presented an important work of interest to people in the field and is worthy of indexing and further working on.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "88748", "date": "11 Nov 2021", "name": "Ahmad Ashrif A Bakar", "expertise": [ "Reviewer Expertise Photonics sensing devices", "surface plasmon resonance", "optical feedback interferometery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 demonstrates the enhancement of stimulated Brillouin scattering in optical microfibers using a numerical model vectorial approach. The Brillouin shift by stimulated Brillouin scattering in a tapered silica fiber of different core and clad radii were investigated. I would suggest the authors have some amendments to improve the quality of the article. The comments are as follows.\nWould you please put more insight into the results and discussion? The explanation of the results is too brief.\n\nI'd recommend the authors to put more discussion in Figure 9 as well. It'd be interesting to see why the graphs are linear in the first stage and gradually saturated once the core diameter reaches 2μm and above.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "7757", "date": "17 Feb 2022", "name": "Hairul Azhar Abdul-Rashid", "role": "Author Response", "response": "Thank you for the comments made to the manuscript and questions raised. We have added to the discussion as follows: Figure 9 demonstrates the relationship between n eff and silica microfiber diameter. n eff in this case here can be seen increasing as the core diameter of optical fiber increases. The n eff value increases ranging from 1.390115 to 1.446122 for 1 μm to 6 μm core diameter of the optical fiber. The values are generated based on the optical model developed in COMSOL. Based on the findings observed, the effective refractive index neff starts to no longer fall within the core and clad refractive index window for uniform microfibers that have core diameters 6 µm and below. This is due to the fact that modes propagating in the microfiber are no longer guided by the core and clad interface but by the air-cladding interface. It becomes more and more pronounced as the core diameter decreases. From the results obtained and shown in Table 4 based on the acoustic model simulations, the smaller core and clad radius of an optical fiber will produce a lower Brillouin shift frequency compared to an optical fiber with a larger core and clad radii. As for the acoustic velocities observed in the optical fibers, a smaller core and clad radii produces a higher acoustic velocity. The Brillouin Frequency shift tabulated in table 4 is obtained from the fundamental eigenvalue based on the numerical model results obtained from the COMSOL model. The Brillouin frequency shift is due to the optical-acoustic interaction. The overlap integral as stated in equation 4 demonstrates the overlap ratio between the acoustic and optical mode in the optical fiber. Shown below are the overlap ratios with respect to their core diameter. The overlap ratios are obtained by solving the overlap integral equation and are tabulated in Table 5 From the results in Table 5, the overlap ratio is influenced by the core and cladding diameter and subsequently affect the Brillouin gain and frequency shift. This is clearly due to the optical mode and acoustic mode profile influenced by the core and cladding diameter. Based on our simulations, a typical silica uniform microfiber with parameters as mentioned above would observed a Brillouin Frequency shift around the 6GHz window. The Brillouin frequency shift for the individual modes namely shear, longitudinal and hybrid were observed to occur at the lower end of the 6GHz spectrum for microfibers that have a smaller core and clad dimensions. As the core and clad dimensions’ increase, we observe the Brillouin Frequency shift to occur at a higher frequency in the 6GHz spectrum. The numerical model therefore helps provide insights into fiber sensor development depending on the sensing frequency of interest. A higher acoustic velocity observed in smaller core and clad fibers also increase the overlap factor of the fundamental modes respectively. In fiber sensors, exposure to external perturbations like temperature will in effect change the acoustic velocity and the Brillouin shift frequency. The Brillouin gain coefficient values can be calculated by substituting the values obtained from the numerical model into the equation as denoted by Equation 5. Table 6 shows the Brillouin gain coefficient for the 2 optical fibers that were modeled. Between the 2 fibers modelled using the numerical model, it was observed that the optical fiber core and clad dimensions of 2 and 6 µm respectively has a higher Brillouin gain coefficient. The higher Brillouin gain coefficient is attributed to the higher overlap ratio, as shown in Table 5. One can relate this understanding to optimize the design of Brillouin amplifiers and sensors with the appropriate Brillouin gain coefficient. Thus, the numerical model here provides insights to the sensor design based on the requirements needed (25)." } ] } ]	1	https://f1000research.com/articles/10-521
https://f1000research.com/articles/10-484/v1	17 Jun 21	{ "type": "Research Article", "title": "Development and psychometric testing of the Thai impact and burden of care scale for caregivers of persons with schizophrenia and co-occurring methamphetamine use", "authors": [ "Ek-uma Imkome" ], "abstract": "Objective: The objective of the present study was to develop the Thai version of the Impact and Burden of Care Scale for Caregivers of Persons with Schizophrenia and Co-occurring Methamphetamine Use (TIBSCSM) and test its psychometric properties. Methods: This instrument development research subjects were 142 caregivers of persons with schizophrenia and co-occurring methamphetamine use. Sample size adequacy was tested by Kaiser–Meyer–Olkin (KMO), and Bartlett's test of sphericity tested the adequacy of the item correlation matrix. The second-order confirmatory factor analysis (CFA) was used to test the theoretical model. Results The 32-item TIBSCSM showed convergent validity correlations with two quality-of-life measures. Additionally, KMO=0.9, Bartlett’s Test of Sphericity χ2=5248.5, df=496, p<0.001, and internal consistency reliability was high (α=0.9). The CFA has shown that the findings are supported by the theoretical models (χ2=325.2, df=287, p<0.001, RMSEA=0.0, CFI =0.9). Conclusion: The TIBSCSM scale has potential benefits for psychiatric nurses and psychiatric care teams to measure the impact and care burden of caregivers of persons with schizophrenia and methamphetamine use in the areas of nursing, research, education, and clinical determination. The test results suggested that The TIBSCSM scale has potential benefits for psychiatric and mental health care team to assess the impact and burden care of schizophrenic caregiver for both research and clinical purposes, especially during the COVID-19 pandemic for providing care to relieve the impact and burden of care.", "keywords": [ "Schizophrenia", "methamphetamine", "caregivers", "factor analysis", "burden care." ], "content": "Introduction\n\nSchizophrenia with co-occurring methamphetamine use is an incapacitating and complex psychiatric illness with either stage, phase, personal factors, and continuous evolution, resulting in impaired physical and psychological health status and social problems. Additionally, it impacts on caregivers’ role and functioning that add to the burden of care and economic consequences.1-3 Moreover, care burden can lead to a low quality of life (QOL), particularly when suffering significant burdens, resulting in role confusion, poor caring performance, psychosomatic problems, anxiety, stress, and depressive symptoms. The negative emotional experience of caregivers may lead to low performance of caring and have crucial adverse effects on medication compliance, continuity, and a good understanding of the care and social support.1,4\n\nThe caregivers work out ways to take care of persons with schizophrenia and methamphetamine misuse with severe psychotic symptoms to the best of their ability. They try to decrease warning signs such as aggressive and violent behaviors as soon as possible. The caregiver tries to give the reasons, call their name, suggest cool water to drink, or shower and express their love and care by speaking gently. They also deal with hardships in caring for patients, which happen to their belongings, other people, and themselves due to harm resulting from psychotic symptoms. During the relapse phase, caregivers work hard with relapses using many ways to prevent injury, set a safe environment, continue medication adherence and soothe their relatives’ psychological state. Additionally, the caregiver tries to pull their relatives back to a normal state as much as possible by encouraging their relatives’ memories and ability to perform activities of daily living.\n\nNumerous studies of evidence-based practice have illustrated that a lack of primary caregiver involvement in treatment planning is related to the treatment adherence issue. Consequently, evaluating the caregivers’ impact and care burden is of considerable relevance both for caregivers and indirectly for the quality of life of persons with schizophrenia with co-occurring methamphetamine use.\n\nThe caregivers’ impact, coping strategies, stress, burden, anxiety, caregiving, and views on the grounds and magnitudes of psychiatric illnesses and co-occurring substance abuse require significant attention. Moreover, currently, the development of psychosocial interventions for caregivers is also a considerable concern. There is an essential need for interventions to enhance caregivers’ emotional health and performance programs delivered by nurses and healthcare teams.5 Based on this, it is necessary to develop good psychometric properties to assess the impact and burden of caregivers of persons with schizophrenia with co-occurring methamphetamine use.\n\nAlthough studies have been conducted on specific issues in caregiving, little has been done to explore the impact and burden among caregivers of persons with schizophrenia. Furthermore, no impact and burden scale has been developed explicitly for use with primary caregivers based on the caregivers’ experience.6 Being anchored in an exact conceptual method is needed. Development of a measure of caregivers’ impact and burden could help elucidate the biological and psychological aspects of caregiving, including quality of life, and preserving caregivers’ well-being and aptitude for care. This can help multidisciplinary treatment teams to develop new care strategies for this population. The Thai version of the Impact and Burden of Care Scale for Caregivers of Persons with Schizophrenia and Co-occurring Methamphetamine Use (TIBSCSM) was developed to assess the impact on caregivers caring for patients with schizophrenia. The objective of this study was to develop a scale and test its psychometric properties.\n\n\nMethods\n\nData were collected from the caregivers of people with schizophrenia with co-occurring methamphetamine use at psychiatric hospitals. The inclusion criteria were: (1) having a family member with a diagnosis of schizophrenia or schizoaffective disorder, according to the DSM-V criteria;7 (2) being identified by persons with schizophrenia and co-occurring methamphetamine use as the primary caregiver; and (3) being 18 years of age or older.\n\nOver four weeks, identified inpatients, who met the criteria of a diagnosis of schizophrenia with co-occurring methamphetamine use and were 18–60 years old, were selected. A psychiatric nurse asked them to name their primary caregiver. The researcher asked them if we could contact the primary caregiver. When they agreed, and when the caregiver met the inclusion criteria, the self-report scale was collected and completed by the researcher teams’ caregivers or interviews.\n\nEthical approval for the study was obtained from the Ethics Review Committee for Research Involving Human Research Participants (COA No. 284/2560). The scopes, risks, and benefits of this study for the subjects were explained. Before data collection, written consent was obtained directly from the PCPSs Participation was voluntary, and participant anonymity and confidentiality were guaranteed.\n\nThe data collected included the following:\n\n1. Demographic characteristics of the primary caregivers.\n\n2. TIBSCSM: The self-administered scale that the caregivers completed.\n\nDevelopment of the TIBSCSM occurred in two phases: a) qualitative and b) quantitative. Item generation occurred during the qualitative phase, and item reduction and the validation process happened during the quantitative phase.8 The two steps involved collecting data from different subjects.\n\nItem generation occurred in two steps. First, the content was derived from the questionnaire from face-to-face, semi-structured interviews performed by the researcher. Briefly, discussions addressed the impact and burden of care based on caregiver stress theory, a middle-range theory. Second, the objective was to predict caregiver stress and its outcomes from demographic characteristics, an objective burden in caregiving, stressful life events, social support, and social roles.9 The determined wording of question stems and the range of response options until saturation by 30 caregivers’ interviews. The researcher performed content analysis. Third, the researcher identifies 32 questions from this interview process. These items were answered using a five-point Likert scale, defined as: 1: never/not at all; 2: rarely/a little; 3: sometimes/somewhat; 4: often/a lot; and 5: always/very much.\"\n\n30 caregivers were requested to remark on any part of the scale (i.e., content, wording, response choices) that they considered inappropriate or merited improvement. Ambiguous items and those that were misinterpreted or infrequently answered were withdrawn or rephrased, leading to a preliminary scale that contained 32 items. Lastly, preliminary interviews were conducted with the caregivers to ensure that the scale was a true reflection of the caregivers’ understanding and confirmed content validity. The second round of interviews with caregivers guaranteed its face validity.\n\nThe item reduction process comes from the results of statistical analyses and the steering committee’s expertise. Item response theory and classical test theory conduct by Statistical approaches.10 Both metrological properties and their impact on the final scale’s content, taking into account the items’ meanings, were discussed and then removed items. The researcher retained items to produce the final version of the TIBSCSM in more robust and psychometrically sound solutions. The researcher tested for construct validity, reliability, and some aspects of external validity in the last version.\n\nConvergent validity was evaluated by examining correlations between the TIBSCSM and two measures of caregivers’ quality of life: the Thai version of the Schizophrenia Caregiver Quality of Life Questionnaire (S-CGQoL-Thai Scale) and the Thai version of the World Health Organization Brief Quality of Life Scale (WHOQOL–BREF–THAI). Construct validity defines the construct to be assessed by the scale and measures its construct’s internal structure and the theoretical relationship of its item and subscale scores. It was evaluated using principal component factor analyses with varimax rotation11 to define the number of independent items and dimensions.\n\n\nResults\n\nThe mean age of the 142 participants was 47.1 years old (SD = 8.4). They were predominantly female (73.9%), married (63.4%), and about half had completed at least a bachelor’s degree (50.7%) and worked in agriculture (57.8%). Additionally, almost all of them (83.1%) had insufficient income, were healthy (61%), and had been the caregiver for more than five years (48%). Regarding medical history, about one-third of them (35.1%) used universal healthcare coverage, and more than half of them had no medical illness (61%) (Table 1).\n\nThe TIBSCSM with 32 items showed content validity index = 1. Regarding convergent validity, the TIBSCSM had correlations with the S-CGQoL-Thai Scale and the WHOQOL–BREF–THAI. Factor analysis showed good construct validity, Kaiser–Meyer–Olkin (KMO) = 0.9, Bartlett’s Test of Sphericity, χ2 = 5248.5, df = 496, p < 0.001. Cronbach’s alpha showed high internal consistency reliability (α=0.9). The corrected item total correlation ranged 0.5–0.8. The total variance explained was 64.9%, which is excellent. Also, the extract construct of factor analysis was the following: physical function, self-esteem, role and social enjoyment, and relationship satisfaction (Figure 1).\n\nCorrelation testing between the 32 items found that the correlation coefficient of all 32 questions was in the range 0.3–0.8, and the correlation coefficient of the internal items of each construct were medium to high in the positive correlation with statistical significance at 0.05 (Table 2, Figure 2).\n\nThe first-order factors loading = 0.5–0.8 and the variance of first-order factors loading were explained by a latent variable of second-order factors of the four constructs ranking 66.3% – 74.0%. Besides, factor loading of TIBSCSM, factor score, and R2 range between = 0.6–0.8, of TIBSCSM.\n\nThe second-order factors analysis of measurement model of TIBSCSM show χ2 = 325.2, df = 287, p = 0.001, RMSEA = 0, CFI0.9, TLI = 0.9, SRMR = 0 (Table 3). This analysis shows that the scale fits with the theoretical. Factor loading: completely standardized solution of second-order factors in the high level (Figure 1). The latent variables can explain the variance of construct of physical function, self-esteem, role and social enjoyment, and relationship satisfaction of 97.2%, 99.5%, 97.0% and 92.5%, respectively.\n\n\nDiscussion\n\nThe findings indicate that the TIBSCSM is psychometrically sound and well-suited for assessing caregivers’ impact and burden of care for persons with schizophrenia and methamphetamine misuse. We discuss the psychometric properties testing in the following sections.\n\nFirst, the internal structure, supported by high internal consistency, confirmed that the TIBSCSM measures a multidimensional concept. Cronbach’s alpha was 0.9. This means strong correlations between items within each domain of the scale and between all items in the scale.12-13\n\nSecond, the KMO test and Bartlett test of sphericity showed the adequacy of the item correlation matrix. This reflects that the sample size was appropriate.11\n\nThird, the components’ analysis showed that the factor loadings were 0.6–0.8, which shows that the items had very high effects on the factors. The criteria for choosing an item is that the factor loading should be greater than 0.5. All questions and questions had a value of 0.5–0.8.11 Additionally, the construct validity of the test was verified by factor analysis. Four components could be extracted together with the results from the scree plot graph. The parts of the four factors explained 64.9% of the variance, which shows that the 32 questions substantially explained variations in the levels of the impact and burden of care in Thai primary caregivers of people with schizophrenia who also use methamphetamine, reliably and consistently with the theory of the Caregiver Stress model. Similar to results from other empirical studies,14 the findings illustrate that the predictors of family caregivers’ impact and burden care were a) physical and psychological well-being, b) dependence in performing activities of daily living, c) caring quality, d) burden and everyday life, and e) caring performance.\n\nAdditionally, the TIBSCSM, like the theory of caregiver stress, is based on the Roy Adaptation Model that identifies the caregiver’s response, perceptions, and adaptations to the stress and burden they experience in terms of their social role and how they reduce and cope with stress. Additionally, it was found that caring for people with schizophrenia often causes caregivers to perceive a high burden of care, lose energy, have anxiety and depression, and have worse overall mental health. There is an increased risk of emotional problems such as guilt, anger, and dissatisfaction. Research has found that most caregivers have emotional stress problems.14 Besides, persistent requests for more help cause caregivers to become frustrated. Sometimes the emotions cannot be suppressed. Intense and negative emotions may occur.\n\nMoreover, it has been found that many caregivers with negative cognitive processes and the inability to provide quality care may experience feelings of failure, resulting in negative perceptions of various aspects, such as having no time for family or other activities or inability to care for people with schizophrenia fully. Additionally, consistent with the caregivers’ stress theory,9 effects on the caregiver include reduced physical function, feelings of low self-worth, and reduced role enjoyment and marital satisfaction, which directly affect the carers’ lives. Individual perceptions of their status in life fall under the context of the culture and system definition in a society that lives and relates to goals, expectations, society standards, and other related things. They covered physical health and mental state, degree of independence, social relations beliefs, and relationships that need to be an environment. Besides, goal burden is the most influential factor for caregivers’ stress, such as caring for hours at a time or care that extends for years. This is consistent with the personal data of more than half of the sample who have taken care of patients for more than ten years. If caregivers perceive high levels of stress, they will make ineffective responses. Additionally, the care burden may lead to depression as a direct result of the stressor may affect stress adjustment.\n\nBesides, these responsibilities may affect other aspects of the caregiver’s life, such as interpersonal relationships. Financial conditions are defined as contextual stimuli. Social support, social roles, and life events that cause stress are life-changing conditions that challenge individuals and result in suffering. In the study sample, 83.10% of participants had incomes that were insufficient to cover their living expenses, and 45.45% had stress levels at 51–75 points.\n\nSocial support allows carers to see that they are being cared for. A high level of social support will increase the ability to cope with the burden effects of care. Additionally, social roles are defined as the caregiver’s relationship to the patient, such as being a parent or child. Most of the sample had one of these two roles, with social functions allowing caregivers to express their feelings and release their emotions, making them more likely to deal with the images and effects of care. The sample had an average age of 47 years, which can change the perspective of each person. Older caregivers have more life experiences and more opportunities to use and adjust their coping skills.15-18\n\n\nConclusion\n\nIn conclusion, in the present paper, the findings suggested that the TIBSCSM scale has potential benefits for psychiatric and mental health care teams to assess the impact and burden of caregivers of people with schizophrenia, for both research and clinical purposes. The TIBSCSM adds exciting data oriented toward providing a more global service to those with schizophrenia and co-occurring methamphetamine use and their families. It would be significant to discover the reproducibility of the current results and their sensitivity to alteration. However, the study demonstrated that the scale has good psychometric properties. The research results deliver an innovative, valid, and essential scale that may be valuable in routine practice, clinical research, and education.\n\n\nData availibility\n\nUnderlying data for this article have been restricted for ethical and privacy reasons. Data may be requested by contacting the authors and access will be granted to researchers and reviewers.", "appendix": "References\n\nImkome E-U, Waraassawapati K: Perspectives and Experiences of Primary Caregivers of Individuals with Schizophrenia in Thailand. Issues Ment Health Nurs 2018; 39(10): 858–864. PubMed Abstract \| Publisher Full Text\n\nDijkxhoorn MA, Padmakar A, Jude N, et al.: Understanding caregiver burden from a long-term perspective: The Banyan model of caregiver experiences. Perspect Psychiatr Care. 2019; 55: 61–71. PubMed Abstract \| Publisher Full Text\n\nLiu N, Zhang J: Experiences of caregivers of family member with schizophrenia in China: A qualitative study. Perspect Psychiatr Care. 2020; (1): 201. PubMed Abstract \| Publisher Full Text\n\nYazar MS, Depçe AA, Balaban ÖD, et al.: The impact of alcohol and substance use by male patients with schizophrenia on burden, quality of life, anxiety, and depression levels of primary caregivers. Anadolu Psikiyatri Derg. 2017; 18(1): 5–12. Publisher Full Text\n\nMagliano L, et al.: Effectiveness of a psychoeducational intervention for families of patients with schizophrenia: preliminary results of a study funded by the European Commission. World Psychiat. 2005; 4: 45–49. PubMed Abstract \| Free Full Text\n\nLevene JE, Lancee WJ, Seeman MV: The perceived family burden scale: measurement and validation. Schizophr. Res. 1996; 22: 151–157. PubMed Abstract \| Publisher Full Text\n\nAmerican Psychiatric Association: Diagnostic and statistical manual of mental disorders. 5th ed Arlington, VA: American Psychiatric Publishing; 2013.\n\nCrocker LM, Algina J: Introduction to classical and modern test theory. Fort Worth: Holt, Rinehart, and Winston; 1986.\n\nTsai PA: Middle-range theory of caregiver stress. Nurs Sci Q. 2003; 16: 137–145. PubMed Abstract \| Publisher Full Text\n\nVan der Linden, Hambleton: Handbook of modern item response theory. New York: Springer; 1997.\n\nHair JF: Multivariate data analysis (8th ed.). Prentice-Hall; 2019.\n\nCronbach LJ: Coefficient alpha and the internal structure of tests. Psychometrika. 2010; 16: 297–334. Publisher Full Text\n\nDeVellis RF: Scale development: theory and applications (4th ed.). SAGE. ; 2017.\n\nCaqueo-Urizar A, Miranda-Castillo C, Giraldez SL, et al.: An updated review on burden on caregivers of schizophrenia patients/Una revision actualizada sobre la sobrecarga en cuidadores depacientes con esquizofrenia. Psicothema. 2014; 2: 235. PubMed Abstract \| Publisher Full Text\n\nOng HC, Ibrahim N, Wahab S: Psychological distress, perceived stigma, and coping among caregivers of patients with schizophrenia. Psychol Res Behav Manag. 2020; 211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParabiaghi A, Lasalvia A, Bonetto C, et al.: Predictors of changes in caregiving burden in people with schizophrenia: a 3-year follow-up study in a community mental health service. Acta Psychiatr Scand. 2007; 116: 66. PubMed Abstract \| Publisher Full Text\n\nDi Sarno M, De Candia V, Rancati F, et al.: Mental and physical health in family members of substance users: A scoping review. Drug Alcohol Depend. 2021; 219. PubMed Abstract \| Publisher Full Text\n\nLabrum T: Caregiving for Relatives with Psychiatric Disorders vs. Co-Occurring Psychiatric and Substance Use Disorders. Psychiatr Q. 2018; 89: 631–644. PubMed Abstract \| Publisher Full Text" }	[ { "id": "93086", "date": "13 Sep 2021", "name": "Virya Koy", "expertise": [ "Reviewer Expertise Instrument developer" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEk-Uma Imkome investigated the topic of Development and psychometric testing of the Thai impact and burden of care scale for caregivers of persons with schizophrenia and co-occurring methamphetamine use. You are focusing on the development and assess the psychometric testing the impact and burden of care among caregivers who provide care to schizophrenia and co-occurring methamphetamine use people. The author has tried to work hard to develop a new instrument with some scientific sections. Moreover, you found an instrument composed of four constructs as follows (1) physical function, (2) self-esteem, (3) role and social enjoyment, and (4) relationship satisfaction. The manuscript is certainly well written and attractive, but I have some major concerns about the instrument development process that prevents me to endorse its acceptance at the present stage. I see the main problems with the statistics that could have led to potentially wrong results and, thus, to completely misleading conclusions.\nTitle:\nCurrent Title: Development and psychometric testing of the Thai impact and burden of care scale for caregivers of persons with schizophrenia and co-occurring methamphetamine use.\n\nSuggestion: to develop and assess the psychometric property test on Burdened Care Caregiver Scale-Thai Version for Schizophrenia and Co-occurring Methamphetamine Use.\n\nEnglish:\nEnglish language, grammar, and punctuation needs to be revised.\n\nAim:\nThe objective is not only development but includes assessment of psychometric proper also. The objective of the present study was to develop the Thai version of the Impact and Burden of Care Scale for Caregivers of Persons with Schizophrenia and Co-occurring Methamphetamine Use (TIBSCSM) and test its psychometric properties.\n\nIntroduction:\nThe author needs to use the concept of interest consistently, e.g. 'the burden of are' and 'care burden'\n\nThe author needs to provide readers with the definition of the concept of interest. Your concept of interest is too long, can you make it short and precise?\n\n“The Thai version of the Impact and Burden of Care Scale for Caregivers of Persons with Schizophrenia and Co-occurring Methamphetamine Use (TIBSCSM) was developed to assess the impact on caregivers caring for patients with schizophrenia”. It should be 'was developed and assessed'\n\nThe author needs to provide the reason why you want to develop this new instrument, why don’t you use the previous instrument?\n\nAgain, you need to provide the readers with different instruments that you believe are not applicable in the Thai context.\n\nMethods:\nResearch design: It should be in 2 phases: first, develop what instrument? Second psychometric properties testing. This it will give readers to catch up well.\n\nInstrument development process:\nBased on DeVillis (2016) there are 7 steps to conduct instrument development.\n\nAuthors need to explain each step therefore, readers can learn from your paper.\n\nThe author missed a critical and necessary step “Generating and item pool”. I can’t understand how the author gets items. The interview may not get items for new instrument development.\n\nThe author can use Delphi Technique or literature review to get enough items, I am not sure the interview can get items for a new instrument.\n\nThe most important step, authors need to conduct a pilot test for readability, comprehensiveness, administrative feasibility, and scoring of the measures, and to identify logistical management issues.\n\nMissed preliminary item tryout, it leads to lack of quality instrument for the next step.\n\nYou need to present CVI: Item-level Content Validity Index (I-CVI) AND Content validity index (I-CVI and S-CVI/Ave).\n\nSample:\nThere are 32 items, at least the author needs 160 participants, 142 is lower than the rule of instrument development.\n\nHow did the author recruit 30 subjects for qualitative study and 142 for quantitative approach?\n\nThe sampling technique is important, must be explained.\n\nA psychometric test involves multi-stage sampling process but not present it.\n\nData analysis: It is a must need.\n\nResults:\nMissed presenting the CVI, it is really needed for a new instrument development.\n\nMissed The Principal Component Analysis (PCA) explanation.\n\nThere are 4 constructs (physical function, self-esteem, role and social enjoyment, and relationship satisfaction) but not sure they are representing to the definition of the new instrument, what is impact and burden meant? The items are not reflected about impacts and burden of care.\n\nDiscussion:\nIdeally, the author(s) discussed statistical analysis. Nevertheless, authors missed the discussion of each construct as the main study, authors need to explain each construct, such as physical function, self-esteem, role, social enjoyment, and relationship satisfaction. The discussion can provide readers an understanding about the researcher's perspective because the researcher is the one who knows the meaning of each construct deeply.\n\nLimitations:\nI expected authors may provide limitations, both quantitative and qualitative.\n\n142 sample are lower than the rule of a new instrument development, it needs to be discussed.\n\nImplication for nursing and research:\nThe implication is the vital part that the researcher has to provide the instructions on how to use this new instrument in nursing practice, and it would provide any additional ideas for the following research. For instance, the TIBSCSM is a new tool developed in the Thai context, and it may replicate the studies to evaluate variations in TIBSCSM across settings if needed.\n\nData needs to be re-analyzed based on the review items and generating item pool procedures to confirm results and conclusions, unless the author provides a comprehensively explanation on how to get full items before to proceed next scientific steps.\nA theoretical framework to the author needs to be addressed and guided for the instrument development process. The authors might want to consider also this for discussion.\nLastly, I think that in the scenario generated based on DeVillis (2016) there are 7 steps to conduct instrument development.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "99304", "date": "15 Nov 2021", "name": "Joko Gunawan", "expertise": [ "Reviewer Expertise Nursing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is important and the scale shows a good validity and reliability. Please note that my comments are not critics; just one-man opinion who needs to understand the authors' work.\nI only have one comment for this study. It is unclear to me whether the authors used the same datasets/samples (142 participants) for PCA and CFA. Please explain it clearly.\n\nIf the authors used the same data, I suggest them to remove all results of CFA, just focus on EFA or PCA. We can’t use both EFA/PCA and CFA with the same data. The reason for this is very simple. We explore factor(s) with EFA; on the other hand, we confirm latent structure/s of the scale with CFA. So, if we try to verify the factor(s) we discovered with EFA using the same data, CFA results will most likely give good fit indices because the same data will tend to conform to the structure(s) of the scale which is discovered with EFA.\n\nOr, if they have different dataset in hand, it is better to conduct another CFA with another data analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-484
https://f1000research.com/articles/10-495/v1	25 Jun 21	{ "type": "Data Note", "title": "Cohort study on educational well-being of children of Chinese origin adopted into transracial and international families in the Spanish education system", "authors": [ "David Doncel-Abad", "Pablo Cabrera-Álvarez", "Pablo Cabrera-Álvarez" ], "abstract": "The following dataset focuses on the educational well-being of adopted girls of Chinese origin in Spanish schools. Due to its characteristics, the presence of this group may generate complex interaction dynamics in school; particularly regarding bullying in school linked to factors such as the acceptance of others. These are dynamics, which may indeed condition the educational experience of this social group. Therefore, the aim of creating this dataset is to measure the educational well-being of children of Chinese origin adopted into transracial families in Spain. Although this research was justified by the lack of studies on this social group, we aimed to go one-step further, we also studied the correlation between this social group’s educational experience, and to what extent they show an interest in Chinese culture. As we have written before, we incorporated the concept of Well-Being and we worked with the following indicators: Satisfaction with Life, Social Life, and Bullying in School, Racial Bullying, Personal Identity and Interest in Chinese Culture. To achieve the objective set forth, we conducted a questionnaire. The final sample consisted of 268 individuals. The creation of this dataset provided us with information that can shed light on the relationship between adoption, race/ethnicity and the educational experience of adopted children of Chinese origin.", "keywords": [ "Well-being", "transnational adoption", "race and ethnicity" ], "content": "Introduction\n\nThe following study focuses on the social well-being or educational experience of adopted girls of Chinese origin in Spanish schools. At the end of the 1990s, Spain became the world’s largest adoption receiving country in the world. Geographically, Asia, and specifically China, was the prefered place among adoptive parents. According to data from the National Statistics Institute of Spain, the first girls adopted from China began to arrive in 1995. From that moment on, Chinese children soon became the most adopted of all: in 2005, out of 2,854 adopted children of Asian origin, 2,753 came from China. A decade later, it is estimated that there are approximately 18,000 adopted children of Chinese origin in Spain (around 400 in Castilla y León), which makes this the largest group when it comes to international adoptions.\n\nThis social group presents a characteristic, which is typical of adoptive families who are of dual nationality and ethnicity: on the one hand, they have Asian phenotypic traits from birth; yet on the other, they are part of the dominant culture due to adoption (Grotevant & VonKorff, 2011). This characteristic, named “the transracial paradox” by Lee (2003), has contributed to the enrichment of social heterogeneity in the classrooms but has also increased the complexity of the relationships formed in the schools (Kim & Lee, 2020). We must not forget that a key element to achieving a necessary and proper coexistence lies in the development of the relationships between the different members of the educational community.\n\nScientific literature on this matter has indicated that one factor linked to bullying in school is racial/ethnic prejudice (Verkuyten & Thijs 2001, 2002, 2006). In particular, a correlation between Asian race or ethnicity and bullying in school has been identified by Juvonen, Grahan and Schsuter (2003) and Mouttapa, Valente, Gallaher, Rohrbach and Unger (2004). Admittedly, it is also indicated that racial/ethnic factor alone does not increase the likelihood of being victimized (Díaz Aguado et al., 2013), yet it is an influencing factor when linked to an array of material and/or social circumstances.\n\nFew studies have been carried out on the analysis of racial/ethnic bullying in school together with the adoption factor in the case of transracial adoptive families. However, there have been significant contributions. Raaska et al. (2012) found that being adopted increases the chances of being bullied in school, but only when combined with other individual variables. Moran et al. (1993) proved that the “being Asian” factor has an impact when it comes to friendship or companionship compared to other social groups. As for the studies carried out by Adams, Tessler and Gamache (2005) and Tang and Arthur (2012), it was proven that if an educational environment offers a positive experience to adopted individuals belonging to transracial families, this contributes to the child’s positive acceptance of the uniqueness of their ethnic or racial origin, fosters their self-esteem and, consequently, their educational well-being. In Spain, Fernández (2016) pointed out that “being Chinese and adopted becomes ‘something bad’ and leads to insults or even bullying” (Fernández-Cáceres, 2016, p. 409). However, Gil, Doncel, Morales and Lambea (2020) revealed that these children may be mocked due to their Asian phenotypic traits, yet not for being adopted.\n\nIn summary, the group of children of Chinese origin adopted in Spain can be considered a group whose presence in schools may generate complex interactional dynamics. Particularly regarding bullying in school linked to factors such as the acceptance of the “other”. These are dynamics, which may indeed condition the educational well-being of this social group.\n\nConsequently, this study aimed to create a dataset that presented the educational well-being of adopted children of Chinese origin in transracial families in Spain, placing special focus on Castilla y León. Although this research was justified by the lack of studies on this social group, we aimed to go one-step further and we also studied the correlation between this social group’s educational experience and to what extent they show an interest in Chinese culture.\n\n\nMethods\n\nThe initial target population of study was formed of female teenagers adopted from the People’s Republic of China who were enrolled in Castilla y León schools. The initial age range was between 14 and 16 years (both ages included). All these girls must have arrived in Spain around 2003-2005, as the data collection started on the 2019-2020 school year.\n\nAccording to the data offered by the Directorate-General for Social Policy, Families and Childhood, under the Ministry of Health, Social Policy and Equality (2010, p. 108), there were a total of 7,612 adoptions from China between 2003 and 2005, which makes this country the most notable when it comes to adoption numbers. Also, taking into account the fact that most adopted children are girls (around 97% according to the data from Castilla y León; Fernández Cáceres, 2016; SSCYL, 2018), we could estimate that the number of girls of Chinese origin was around 7,380. The census carried out between 1997 (the year when the city of Burgos started receiving the first girls from China) and January of 2020 registered a total of 820 adoptions in Castilla y León. Out of these 820 adoptions made in the People’s Republic of China, 800 were girls (97.6%), and 20 were boys (2.4%) (SSCYL, 2018; Fernández Cáceres, 2016). The strict birth control measures and the one-child policy implemented in the People’s Republic of China, together with the patriarchal tradition of preferring sons to daughters, explains why girls were abandoned more than boys.\n\nAs we were lacking a sampling framework, we decided to ask for cooperation from adoptive parents’ associations to be able to access the target population. We collected data through a census, which showed parents who had joined adoption associations and who were able to complete an adoption process in China between 2003 and 2006. This approach is justified by the impracticability of screening the general population in order to locate homes with adopted children. Furthermore, this strategy is based on the evidence that a large number of parents who adopt children from China tend to join an association in order to go through this process with greater guarantees.\n\nThis strategy allowed us to access a large group of adopted children through their parents. In order to do so, we created an exhaustive list of adoptive parents’ associations registered in Spain: ADOPCHINA, AFADA, AFADENA, AFAAR, AFAHU, AFAIC, AFAMUNDI, AIBA, AMADA, ANICHI, ARFACYL, ASTURADOP, ATLAS, AKUNA INTERNACIONAL, ACI, Hijos que esperan, MANANIA, MUDAN, UME ALAIA, TRANSMES, Fundación CORA and AFAC. The research team requested these associations to forward our study to their members who met the requirements to be a part of the target population for this research.\n\nThe online questionnaire addressed to adopted children was sent by email to the contacts provided. We first sent the questionnaire on September 12, 2019 at 12:00 AM. The responses to the questionnaire were gathered over three months (from October 2 to December 17). While compiling the responses, we exhaustively monitored the sample and the established exclusion criteria.\n\nThe incidents we encountered while gathering the responses to the questionnaire were as follows:\n\n• A questionnaire was deleted because it was duplicated.\n\n• Two questionnaires were deleted because they were incomplete.\n\nOn December 17, we obtained the final sample, which consisted of n = 268 individuals between 9 and 19 years, both ages included. Of them, 98.1% were girls and 1.9% boys.\n\nIt is worth noting that there could have been some measurement bias due to the children filling in the questionnaire under their parents’ supervision. It is possible that the children felt inhibited or that their responses were not as precise because of their parents being present.\n\nIn this study, we adopted the concept of Well-Being used by the Organization for Economic Cooperation and Development (OECD) in its PISA 2017 report in order to measure the educational well-being of this social group in schools. The OECD related the concept of students’ social well-being to a variety of factors: satisfaction with life, schoolwork-related anxiety (homework and tests), motivation to achieve, sense of belonging at school, bullying, and social status and inequalities. Taking into account these elements influencing the concept of Educational Well-being, and in accordance with the purpose of this study, we have looked at the following factors: Satisfaction with Life, Social Life, Bullying in School, Racial/Ethnic Bullying, Personal Identity and Interest in Chinese Culture.\n\n• Satisfaction with Life measures the degree of subjective well-being of students in relation to their own lives. In the questionnaire used in this study, the interviewees’ satisfaction was rated on a scale of 0 to 10, 0 being the lowest satisfaction and 10 the highest.\n\n• Social Life (α = .81) has been defined as social integration or social relationships that the individual has established with his or her classmates. The items used to measure the degree of social integration were: “We get along well in the classroom,” “I express and defend my opinions without hurting others,” “I feel that I have friends,” “My classmates care about me when I need them,” “I like to collaborate with my friends,” “My classmates like me,” “I’d rather work in teams than alone,” “I felt different from the rest,” “It is hard for me to express my feelings.” Each indicator was rated on a Likert scale, ranging from 1 (Strongly Disagree) to 5 (Strongly Agree). Based on these indicators, we created the Social Life Scale, the scale being from 1 (low degree of social integration at school) to 5 (high degree of social integration).\n\n• Bullying (α = .72) has been defined as “an intentional, repeated, negative (unpleasant or hurtful) behavior by one or more persons directed against a person who has difficulty defending himself or herself” (Olweus, 2006, p. 81). In this research, this scope was studied by asking the typical questions to measure bullying in PISA: “I have been physically hurt by other students,” “Other students took my things from me,” “Other students insulted me/called me names,” “Other students lied about me, talked behind my back or spread rumors about me,” “I was threatened by other students,” “Other students left me out of things/isolated me on purpose,” “Other students destroyed my things” and “Other students made fun of me.” Each indicator was measured on a Likert scale (1-Never, 2-Rarely, 3-Sometimes, 4-Frequently and 5-Always). From these indicators, we created the Bullying Scale, ranging from 1 (low perception of having experienced bullying) to 5 (high perception of having experienced bullying).\n\n• Racial/Ethnic Bullying was studied by adding a racial element to the typical questions to measure bullying: “I felt intimidated, rejected or bullied because of my facial features.” This indicator was measured on a Likert scale (1-Never, 2-Rarely, 3-Sometimes, 4-Frequently and 5-Always).\n\n• Personal Identity: In order to measure children’s perception of identity, we used one of the Social Life indicators: “I felt different from others.” This item was measured on a Likert scale, 1 being Strongly Disagree (greater integration) and 5 being Strongly Agree (little integration).\n\n• Interest in Chinese Culture (α = .88), measured by the following indicators: “News about China interests me,” “I’d like to learn Chinese,” “I’d like my parents to go to Chinese restaurants more often,” “I’d like to attend Chinese celebrations,” “I’d like to know how non-adopted Chinese children live in my town,” “I’d like my parents to take me to China” and “I’d like my school to organize activities so I can learn about Chinese culture.” Each item was measured on a Likert scale, 1 being Strongly Disagree and 5 Strongly Agree. Based on these indicators, we created the Interest in Chinese Culture Scale, the scale ranging from 1 (little interest in learning about Chinese culture) to 5 (strong interest).\n\nWe added a set of sociodemographic variables to the analysis, these being:\n\n• ISCED: Scale to measure the parents’ highest completed level of education, 1 being completed secondary education or lower and 5 being university education (master’s degree or doctorate).\n\n• Age: recoded in four intervals: 9-13; 14-15; 16-17 and 18-19 years old.\n\n• Age of arrival in Spain, measured in months.\n\n• Type of school: Público (Public School), Concertado (Charter School), Privado (Private School)\n\n• Sex.\n\n\nComposition of the final dataset\n\nThe final sample (n = 268) consisted of a larger number of participants than expected from the initial sample design. Also, a broader part of the Spanish territory was represented. However, the sample criteria established for Castilla y León, which was the region we were interested in, were not met. We obtained data from adopted children of Chinese origin from the following Autonomous Communities (see Table 1):\n\nIn the following table (Table 2), we can observe the profile of the final sample by sociodemographic variables:\n\n\nSummary\n\nThe main results obtained show:\n\n• The incidence of bullying in school is similar to the parameters observed in other reports (like PISA 2015) concerning the school population in Spain.\n\n• Moreover, this group stands out in particular due to its high level of Satisfaction with Life (in relation to expected results by age group compared to data from the PISA 2015 report for Spain) and its low perception of feeling different from others.\n\n• The fact that there was not a defined sampling framework that the researchers could access led them to use associations as intermediary actors in order to distribute the questionnaire among the target population of study. This lack of a sampling framework implied that many adopted children meeting the criteria for the target population were not included in the sample because their parents did not belong to an adoption association.\n\n• Due to the difficulties in accessing such a specific population, we created a census from the lists of members of the Spanish associations of parents who adopted their children in China. When generalizing the results, we must take into account that not all adoptive parents are part of an association and that some of the contacted parents declined to respond. These two sources of bias (coverage and nonresponse) are common to most surveys.\n\n\nEthical approval\n\nSince the sample would be partially made up of minors, we requested the approval of the Bioethics Committee from the University of Salamanca. After checking that an ethical collection of minors data was guaranteed, the Bioethics Committee approved this project (ID: 390) on September 30, 2019.\n\n\nConsent\n\nTo carry out this project, before collecting the questionnaires, we obtained the informed consent form prior to the participants or their parents, in cases of minors, taking part in the research.\n\n\nData availability\n\nThe data collected for this study cannot be made available to the public in an open repository since the parents, who consented before the children responded to the questionnaire, were informed that the data would be encoded, anonymized and stored in the Archives of the Faculty of Social Science of Salamanca—and that they would only be used for the purposes of this project. It was also agreed that, if any other researchers wished to access this database, they would have to contact the Faculty of Social Science of the University of Salamanca.\n\nThis project’s database is stored in the Archives of the Faculty of Social Science of the University of Salamanca, located in Campus Unamuno s/n, Salamanca 37006, Spain. This space can only be accessed by the members of the Deanery and the University Secretariat. If a third party wished to access this material, they would need to ask the Archives authorities for the corresponding authorization. Contact details: davidoncel@usal.es Telephone 923294500 Ext. 3113. All the information derived from this study will be used exclusively for the purposes indicated in the initial project.", "appendix": "Acknowledgements\n\nThis database is part of the Research Project SA157G18 (J425-463AC03), entitled “Antropología transversal del conocimiento: Castilla-León y Asia Oriental”, developed by the accredited research group Humanismo Eurasia from the University of Salamanca. Funding Entity: Junta de Castilla y León. This article is based on a previous study entitled “Study on Child Well-being of Adopted Chinese Children at Schools in Castilla y León”. Source: Pedagogía Social. Revista Interuniversitaria, Issue 35, p. 81-93. DOI:10.7179/PSRI_2019.35.06. It is also based on the article “Educational Well-being and Social Identity of Children of Chinese Origin Adopted in Spain”, which is currently being reviewed by the Adoption Quarterly journal.\n\n\nReferences\n\nDíaz-Aguado J, Martínez Arias R, Martín Babarro J: El acoso entre adolescentes en España. Prevalencia, papeles adoptados por todo el grupo y características a las que atribuyen la victimización: Bullying among adolescents in Spain. Prevalence, participants’ roles and characteristics attributable to victimization by victims and aggressors. Ministerio de Educación; 2013.\n\nFernández Cáceres MI: Familias castellanoleonesas adoptantes en China, 1995-2015: decisión de adoptar, relaciones familiares y estrategias de conciliación. Tesis Doctoral. Salamanca:Universidad de Salamanca; 2016. Publisher Full Text\n\nGil Villa F, Doncel Abad D, Morales Romo N, et al.: Percepción del bienestar social de niños y niñas adoptadas de origen chino en las escuelas en Castilla y León. Pedagogía social: revista interuniversitaria. 2020; 35: 73–85. Publisher Full Text\n\nGrotevant HD, Von Korff L: Adoptive identity. In: Handbook of identity theory and research. New York, NY:Springer; 2011; (pp. 585–601).\n\nJuvonen J, Graham S, Schuster MA: Bullying among young adolescents: the strong, the weak, and the troubled. Pediatrics. 2003 Dec; 112(6 Pt 1): 1231–7. PubMed Abstract \| Publisher Full Text\n\nKim AY, Lee RM: A critical adoption studies and Asian americanist integrative perspective on the psychology of Korean adoption. In: Miller G, Helder E, Marr E. (editors) The Routledge Handbook of Adoption, Routledge International Handbooks. 2020; pp. 120–134.\n\nLee RM: The transracial adoption paradox: History, research, and counseling implications of cultural socialization. Couns Psychol. 2003; 31(6): 711–744. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinisterio del Interior: Sistema Estadístico de Criminalidad.2016. Reference Source\n\nMoran S, Smith PK, Thompson D, et al.: Ethnic differences in experiences of bullying: Asian and white children. British J Edu Psychol. 1993; 63(3): 431–440. Publisher Full Text\n\nMouttapa M, Valente T, Gallaher P, et al.: Social network predictors of bullying and victimization. Adolescence. 2004; 39(154), 315. PubMed Abstract\n\nOECD: PISA 2015 Results (Volume III): Student´s Well-Being. París OECD; 2017.\n\nRaaska H, Lapinleimu H, Sinkkonen J, et al.: Experiences of school bullying among internationally adopted children: Results from the Finnish Adoption (FINADO) Study. Child Psychiatry Hum Dev. 2012; 43(4), 592–611. PubMed Abstract \| Publisher Full Text\n\nTan TX, Jordan-Arthur B: Adopted Chinese girls come of age: Feelings about adoption, ethnic identity, academic functioning, and global self-esteem. Children and Youth Services Review. 2012; 34(8): 1500–1508. Publisher Full Text\n\nThomas KA, Tessler RC: Bicultural socialization among adoptive families: Where there is a will, there is a way. J Family Issues. 2007; 28(9): 1189–1219. Publisher Full Text\n\nSSCYL (Servicios Sociales de Castilla y León): Boletines informativos anuales sobre adopción.1999-2018Reference Sourceconsulta en junio de 2018.\n\nVerkuyten M, Thijs J: Peer victimization and self-esteem of ethnic minority group children. J Community y Appl Soc Psychol. 2001; 11(3): 227–234. Publisher Full Text\n\nVerkuyten M, Thijs J: School satisfaction of elementary school children: The role of performance, peer relations, ethnicity and gender. Soc Indicators Res. 2002; 59(2): 203–228. Publisher Full Text\n\nVerkuyten M, Thijs J: Ethnic discrimination and global self-worth in early adolescents: The mediating role of ethnic self-esteem. Int J Behav. Develop. 2006; 30(2): 107–116. Publisher Full Text" }	[ { "id": "100287", "date": "29 Nov 2021", "name": "José David Urchaga-Litago", "expertise": [ "Reviewer Expertise Methodology and Statistics in Social and Human Sciences" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract: Studies the incidence of bullying in school and satisfaction with life in a sample of children adopted from China.\n\nThis study is very new in Spain. In addition, it is very important because adopted children of Chinese origin is the largest group.\n\nIt is original to study “to what extent they show interest in Chinese culture”, but it is important to justify why that is, to briefly justify its theoretical importance. Neither is any result provided on this aspect. In this case, either present results or eliminate said objective (I recommend the latter).\n\nWell defined population and sample. Well measured variables. The authors are requested to reference the original studies of the measuring instruments. If they have been created \"ad hoc\" for this research, the authors must indicate it.\n\nTable 1: Eliminate the accumulated percentage.\n\nImportant: There is no Results section. Provide the study data about these conclusions:\nThe incidence of bullying in school is similar to the parameters observed in other reports (like PISA 2015) concerning the school population in Spain.\n\nMoreover, this group stands out in particular due to its high level of satisfaction with life (in relation to expected results by age group compared to data from the PISA 2015 report for Spain) and its low perception of feeling different from others.\nI do not know if the journal admits publishing studies without results, only presenting the conclusions. If so, I apologize for the last entry.\nI consider this to be a very important and original study. I think it should be indexed.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "7816", "date": "17 Feb 2022", "name": "David Doncel-Abad", "role": "Author Response", "response": "First, we want to thank the reviewer for his useful comments that have been fundamental to refining the final version of the paper. Moreover, we would like to add some clarifications to some of your questions and highlight the changes introduced in the new version of the paper. We adapted the statement about the research objectives, which are broader than assessing to what extent “the Chinese adopted children show interest in Chinese culture”. This secondary objective will be developed and assessed in other publications in the frame of the research project. We understand that this statement could be misleading, and we have removed it from the paper. We have included the references of the measures used in the study. Regarding the absence of a results section: This is a data note in which we describe a dataset and inform about the conditions under which other researchers can access it. The substantive results of this research are presented in other papers that are under review at the moment. However, we agree with the reviewer that the comments about the conclusions should not be part of a data note, and, therefore, we have removed them from the paper. We improved the paper with further clarifications in the methods section and style changes." } ] }, { "id": "100286", "date": "11 Feb 2022", "name": "Kwok Ng", "expertise": [ "Reviewer Expertise public health and health promotion." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nicely reported data set with rationale and background to the sample. Furthermore, given the limitations of the sampling frame, the authors have done a good job to locate such a diverse range of participants with similar characteristics of interest. The data availability is of course a limitation, but the authors have given information for how to retrieve it, and although it has not been attempted, it is hoped that it would not impede on the progress of open science. The variables are slightly limited, given the efforts taken to collect data from the sample, and a more holistic perspective of health may have been warranted. None the less, this is a starting point to understand the exposure to victimisation and self-perceptions around this matter from the perspective of Chinese adopted girls in Spain.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-495
https://f1000research.com/articles/10-508/v1	28 Jun 21	{ "type": "Research Article", "title": "Cross-sectional assessment of cardiovascular risk factors in patients with knee osteoarthritis", "authors": [ "Sagar Goel", "Surendra Umesh Kamath", "Rajendra Annappa", "Sunil Lakshmipura Krishnamurthy", "Manesh Jain", "Samarth Thakkar", "Lulu Damsas", "Sayak Banerjee", "Prajwal Madapura Divakar", "Sagar Goel", "Rajendra Annappa", "Sunil Lakshmipura Krishnamurthy", "Manesh Jain", "Samarth Thakkar", "Lulu Damsas", "Sayak Banerjee", "Prajwal Madapura Divakar" ], "abstract": "Background: Osteoarthritis (OA) and cardiovascular disease (CVD) are prevalent in India. However, there is dearth of literature among Indians studying the relationship between the two. This study was carried out to assess various cardiovascular (CV) risk factors in patients with knee OA with an objective to investigate their association, screening and management. Methods: In total, 225 patients were included in this cross-sectional study. Participants were diagnosed with knee OA on the basis of the Kellgren and Lawrence (K-L) classification of their radiograph. Participants were also assessed for CV risk factors (age, body mass index, systolic blood pressure, diabetes mellitus, total cholesterol, high-density lipoprotein, smoking) with the help of the Joint British Society QRisk3 calculator (JBS3), which gave three variables: JBS3 risk score, heart age, and life expectancy. Chi Square, Fishers exact test and one-way ANOVA tests were used to compare the categorical and quantitative variables, respectively. Pearson’s correlation coefficient was used to assess the relationship between CV risk factors and knee OA. Results: Patients with severe knee OA had a statistically significantly higher prevalence of CV risk factors (p<0.05). Grade 4 knee OA patients were found to have a mean JBS3 risk of 38%, heart age of 82 years and life expectancy of 77 years as compared to grade 2 patients who had a mean JBS3 risk of 11%, heart age of 63 years and life expectancy of 82 years. Conclusions: Our study concluded that there is a strong positive correlation between knee OA and CVD, with CV risk score being directly proportional to the severity of OA. JBS3 is a comprehensive risk score calculator as well as a screening tool, which produces three more comprehensive variables, namely 10-years risk of developing CVD, physiological heart age and life expectancy.", "keywords": [ "Osteoarthritis", "Cardiovascular disease", "Cardiovascular risk factors", "JBS3 risk", "Hypertension", "Diabetes Mellitus", "Smoking", "Age", "Sex", "SES", "Heart Age", "Life Expectancy", "Kellgren Lawrence" ], "content": "Introduction\n\nOsteoarthritis (OA), a degenerative joint disease, is the sixth most predominant cause of disability around the world and a well-established cause of restricted activity, disability, and low quality of life.1 Its prevalence, currently at 3.5% of the world’s total population and about 35% in adults older than 60 years2, is on the rise. OA has become an important public health issue and is burdensome for both personal health and social well-being.2 Both developed and developing nations see it as a major liability on their health care sector with it having severe social and economic impacts.1\n\nOA is associated with knee pain, functional limitation and deformity and it incurs articular cartilage degeneration, osteophyte formation in joints, reduced joint space and subchondral sclerosis.3 The etiology of OA is idiopathic, yet age, genetics, obesity, menopause, hypertension, and diabetes mellitus have been found to be major contributors.4 Some of these risk factors that are directly involved and others indirectly involved in the etiopathogenesis of OA are also seen to increase the risk of cardiovascular diseases (CVDs), namely congestive heart failure (CHF), ischemic heart disease (IHD), transient ischemic attacks (TIA) and stroke.4 This group of cardiovascular diseases are also the leading cause of mortality and morbidity worldwide.5 Therefore, it becomes important to identify the various cardiovascular risk factors like age, gender, obesity, hypertension, cholesterol, sedentary lifestyle, smoking and nutrition at the earliest opportunity through various screening methods, especially in patients with OA.3\n\nAn interest in the association between OA and cardiovascular risk factors is increasing as recent studies have reported that OA is associated with higher blood sugar and serum cholesterol levels in females as compared to healthy females.3,4 Singh et al. also found a high prevalence of cardiovascular risk factors in adults with knee OA.5 Recently, a new classification for phenotyping of OA, which includes metabolic syndrome, aging, and posttraumatic arthritis, has been developed. Obesity is a well-known contributor for metabolic syndrome.6 Observational studies have shown that hypertension is an independent risk factor for knee OA.6,7 Systemic inflammation is also a risk factor for CVD.8 Indirectly, physical inactivity, due to debilitating joint pain, also makes patients more prone to CVD.7\n\nGiven that OA and CVD are common conditions in the elderly, a thorough knowledge of the relationship between OA and CVD could help further our understanding of potential biological and behavioral mechanisms, which could in turn help in making informed decisions regarding further management of OA. There is a dearth of literature relating knee OA and cardiovascular risk factors in the Indian population, hence we wanted to study this association among Indian participants. The objectives of the study were (i) to screen patients diagnosed with knee OA for cardiovascular risk factors who don’t have a history of cardiovascular diseases; (ii) to find out the risk of developing a cardiovascular disease in next 10 years in patients suffering from knee OA using a simple standardized risk score calculator; and (iii) to correlate CVD risk with severity of knee OA.\n\n\nMethodology\n\nThe research protocol with the informed consent form was approved in October 2018 by the Institutional Ethics Committee of Kasturba Medical College, Mangaluru (reference number IEC KMC MLR 10-18/363). Participants provided written informed consent to participate. In addition, case records of patients diagnosed with knee OA between January 2017 to September 2018 were studied. Permission to access the previous records was obtained from the medical suprintendant of the hospitals associated with Kasturba Medical College, Mangalore through the proper channel including head of department of orthopedics and Institutional Ethics committee. Confidentiality of the participants was maintained by concealing their name and identity. Each participant was given a unique serial number. The patients were not charged extra for any test or investigation other than their routine healthcare charges.\n\nThis cross-sectional study was done in the tertiary care centres associated with Kasturba Medical College, Mangalore, India. The study was carried out between October 2018 and September 2020.\n\nPatients diagnosed with knee osteoarthritis (Kellgren and Lawrence (KL) grade ≥2 on knee X-ray11) and ≥50 years old9,10 were invited to take part. The KL grading system is as follows:\n\n1. Grade 0 (none): the definite absence of x-ray changes of osteoarthritis\n\n2. Grade 1 (doubtful): doubtful joint space narrowing and possible osteophyte lipping\n\n3. Grade 2 (minimal): definite osteophyte and possible joint space narrowing\n\n4. Grade 3 (moderate): moderate multiple osteophytes, definite narrowing of joint space, and some sclerosis and possible deformity of bone ends.\n\n5. Grade 4 (severe): large osteophytes, marked narrowing of joint space, severe sclerosis and definite deformity of bone ends.\n\nExclusion criteria were patients who refused to give consent, patients who were suffering from secondary knee OA, and patients who were known cases of coronary artery disease.\n\nThe sample size was calculated to be 187 using the Cochran’s formula12:\n\nwhere, Z∝ = 1.96 at 95% confidence level\n\np (estimated proportion of an attribute that is present in the population) = 34% with respect to study by Kim HS et al.13\n\nq = 66% (q = 100-p)\n\nd (desired precision) = 20% of p at 80% power\n\nWith 95% confidence level and 80% power. Though the sample size was calculated to be 187, we collected data from 225 patients due to additional time and resources.\n\nThis study employed convenience sampling. Patients aged more than 50 years presenting to the orthopedics out-patient department with complains of knee pain between October 2018 and september 2019 were recruited for the study. Also, case records of patients diagnosed with knee OA between January 2017 to September 2018 were studied. No prior randomization was done.\n\nPatient demographics such as age, gender, weight, height, body mass index (BMI), socioeconomic status and their smoking habits were recorded. Weight bearing knee radiographs in anterior, posterior and lateral views were done. Those with knee OA of K-L grade of 2 or more were included in the study. Serum total cholesterol and high-density lipoprotein (HDL) was determined. History of ongoing treatment for hypertension, diabetes, RA and CVD in relatives before age of 60 years was gathered. All these variables were added in the JBS3 risk score calculator which then produced three more variables; physiological heart age, life expectancy, and JBS3 risk of developing CVD in the next 10 years.\n\nJBS3 risk score calculator was devised by joint British society for general practitioners to help guide their work with patients, in preventing CVD. A major role of the JBS3 risk score calculator is the idea of estimating CVD risk over a lifetime. Patients can also be screened for cardiovascular risk factors that they are unaware of.\n\nCase records of patients aged 50 years and above, diagnosed with knee osteoarthritis, between January 2017 and September 2018, and patients coming to the Out Patient Department (OPD) from October 2018 to September 2019 were studied. Patients were briefed about the aims and implications of the study, and consenting patients were recruited into the study.\n\nEach patient was given a unique serial number and a detailed history and examination were done by the principle investigator of the study. History of presenting illness along with past, treatment and family history were gathered. Examination included general physical examination along with cardiovascular systemic examination and local knee examination. Orthopedic evaluation was done using K-L grading11 and CV risk was determined using the JBS3 CV risk score calculator.14 Fasting blood sample was collected to know the levels of their serum total cholesterol and serum high density lipoprotein. The standard procedure of phlebotomy was followed. 3ml of blood was drawn from the patient and collected in plain vacutainer for serum analysis of total cholesterol and serum high density lipoprotein. The sample was sent to Biochemistry laboratory where they analysed using enzymatic colorimetric method using enzyme cholesterol esterase and peroxidase for total cholesterol and polymer polyanion method for HDL. Patient details and blood test results were uploaded to the unique profile created for each one of them in the mobile application based JBS3 CV risk score calculator14 which then predicted physiological heart age, life expectancy, and JBS3 risk of developing CVD in the next 10 years.\n\nThe Statistical Package for the Social Sciences (SPSS) version 20 was used to do the statistical analysis. The categorical variables like age, gender, smoking habits, ongoing treatment for hypertension, diabetes, history of CVD in relatives before age of 60 years and history of rheumatoid arthritis (RA) were compared with K-L grade of knee OA using chi square test. Socioeconomic status and history of RA were compared with K-L grades using Fishers exact test. The quantitative variables like BMI, total cholesterol, HDL, physiological heart age, life expectancy and 10 years risk of developing CVD were compared with K-L grades using analysis of variance (one-way ANOVA) test. A p value of <.05 was considered significant and less than 0.01 was considered highly significant.\n\nPost hoc analysis was done to compare multiple group means. The mean heart age and mean physiological heart age among the three K-L grades of knee OA were compared using paired t test. A p value <.05 was considered significant. Pearson’s correlation coefficient was employed to determine the correlation between the K-L grade of knee OA and quantitative variables like serum total cholesterol (TC), HDL, BMI, SBP, physiological heart age, life expectancy and 10 years risk of developing CVD. Receiver operating characteristic (ROC) curve was used to show in a graphical way the connection/trade-off between clinical sensitivity and specificity for 10 years risk of developing CVD in the study population.\n\n\nResults\n\nA total of 225 patients visiting the orthopedics OPD with complaints of knee pain took part in the study.32 All participants were over 50 years of age with more than half (53%) of them between the age of 50-60 years (Figure 1). The majority of these patients were females (59%) (Figure 2). As per the BG Prasad socioeconomic scale (SES),15 the majority of them belonged to either the middle or lower socio-economic group (Figure 3).\n\n59% of the study population were female, 41% were male.\n\n44% of the study population belong to the lower middle class and 27%, 22%, 5%, and 1% belong to lower class, middle class, upper-middle-class, and upper class, respectively.\n\nGrade 2 K-L classification of knee OA was found to be the most common (45%) among the study population. Higher grades of knee OA, i.e. 3 and 4, were found to be 42% and 13%, respectively (Figure 4). Overall, 57% of the patients belonging to the lower socio-economic class as per the BG Prasad SES had knee OA grade 2, 33% of them had grade 3 and only 10% had grade 4 OA. Among the lower middle and middle-class groups, a decrease in the percentage of patients with grade 2 and increase in percentage of patients with grade 3 and 4 was found as socioeconomic status increased. The same trend was observed, not strictly though, as we go further up the SES. Upper middle and upper-class patients had either grade 3 or 4 knee OA (Figure 5).\n\n45%, 42%, and 13% of the study population had grade 4, grade 3, and grade 2 of K-L classification of knee OA, respectively.\n\nHigher socioeconomic groups had a higher proportion of patients with severe grade knee OA.\n\nThe Fishers exact test was used to find out the statistical significance of the relationship between socioeconomic status and knee OA grade, which was found to be highly significant (p value<.01). Chi square test, when applied to compare knee OA grade with age {X2(4, 225) = 3.801, p = .434} or gender {X2(2, 225) = 1.016, p = .602}, was not found to be statistically significant. A one-way ANOVA test was applied to K-L grade of knee OA with BMI and found to be highly statistically significant {F(4,225) = 60.652, p < .01}. An increasing trend in the BMI of patients was noted with a higher grade of knee OA, e.g. 60% of grade 2 knee OA patients had a BMI of less than 25 as compared to 40% in grade 3 and only 7% in grade 4. In addition, 57% of patients with grade 4 knee OA had a BMI of more than 30 as compared to 13% in grade 3 and only 4% in grade 2 (Figure 6).\n\nParticipants with higher grade of knee OA have higher BMI.\n\nThe mean BMI of patients with grade 2 knee OA was 24.82, grade 3 was 26.39 and grade 4 was 30.96, with each knee OA grade showing statistically significant data when compared with the BMI of patients. In post hoc analysis using the Bonferroni test with BMI as the dependent variable, it was found that multiple comparisons of each of grade of knee OA with the other were found to be statistically significant, as depicted in Table 1.\n\nWith BMI as dependent variable there is significant difference when all the three grades of OA knee are compared with each other. OA = osteoarthritis; HS = highly significant.\n\nOf the 225 participants, 18.2% (n = 41) were smokers. All the smokers were male, and 39% of them had grade 2 knee OA, 44% of them had grade 3 and 17% of them had grade 4. However, this data was not statistically significant on chi square test {X2(2, 225) = 0.964, p = .617}.\n\nThe mean systolic blood pressure (SBP) of participants was 128mmhg, 139mmhg and 150mmhg for grade 2, grade 3 and grade 4 knee OA, respectively. Figure 7 shows the representation of mean SBP for each K-L grade. On comparing each grade with each other using post hoc analysis with SBP as the Bonferroni dependent variable, the comparisons came out to be highly significant (Table 2).\n\nThe mean systolic blood pressure (SBP) was 128mmHg, 139mmHg and 150mmHg for participants with grade 2, grade 3 and grade 4 knee OA, respectively.\n\nWith SBP as dependent variable there is significant difference when all the three grades of OA knee are compared with each other. OA = osteoarthritis; HS = highly significant.\n\nOf the 225 participants, 88 had a SBP reading of 140 or more, out of which 28 (33%) were not taking any antihypertensive medication. In total, 25.8% of the entire sample had a history of diabetes mellitus (DM), which is much lower than more than half (53%) of patients with grade 4 knee OA. Of grade 3 patients, 40% had DM, yet of grade 2, only 4% had DM. This was found to be statistically significant {X2(2, 225) = 47.574, p < .01} as per the chi square test. Figure 8 depicts the comparison between each grade of knee OA and diabetes mellitus.\n\nOverall, 25.8% of all patients gave history of diabetes mellitus (DM) which is much lower as compared to more than half (53%) of patients with Grade 4 OA knee having DM. 40 % of grade 3 patients had DM and only 4 % of grade 2 patients had DM.\n\nSimilar trends were seen in the history of CVD among the relatives of the study population. A total of 35 (15.5%) patients gave a history of their relatives suffering from CVD in the past. Compared by grade, 46.7% of the patients with grade 4 knee OA gave a positive relative CVD history as compared to only 17% and 5% of the patients with grade 3 and 2 knee OA, respectively (Figure 9). The chi square test for this difference was statistically significant {X2(2, 225) = 30.907, p < .01}.\n\n46.7% of the patients with Grade 4 knee OA gave a positive history in this aspect as compared to only 17% and 5% of the patients with grade 3 and grade 2 respectively.\n\nNone of the patients in the study population suffered atrial fibrillation (AF) or gave a history of chronic kidney disease in the past. Only 11 patients gave a history of rheumatoid arthritis which was found to be statistically significant by Fisher exact test (p < .01).\n\nSerum total cholesterol (TC) and high-density lipoprotein (HDL) were tested for all the patients. Figure 10 shows the mean levels of TC and HDL with respect to each of the KL grades of knee OA. An increase in mean value of TC and decrease in mean value of HDL were noted as K-L grades of knee OA increase. Patients with grade 2 knee OA had a mean TC value of 188.34 and mean HDL value of 53.74. Similarly, patients with grade 3 knee OA had a mean TC and HDL value of 213.66 and 51.99, respectively. Patients with grade 4 knee OA had a mean TC and HDL value of 234.90 and 49.30, respectively (Figure 10).\n\nSimilarly, patients with Grade 3 knee OA had a mean TC and HDL value of 213.66 and 51.99 respectively. And patients with Grade 4 knee OA had a mean TC and HDL value of 234.90 and 49.30 respectively.\n\nIn post hoc analysis using the Bonferroni test with TC as the dependent variable, it was found that multiple comparisons of each of grade of knee OA with the other were found to be highly statistically significant (Table 3). The same was not true, however, with HDL, which showed statistically significant data only when K-L grade 2 was compared to grade 4 knee OA (Table 4).\n\nWith serum total cholesterol as dependent variable there is significant difference when all the three grades of OA knee are compared with each other. OA = osteoarthritis; HS = highly significant.\n\nWith HDL as dependent variable there is significant difference when Grade 2 is compared with grade 4 but not when grade 3 is compared with grade 2 or grade 4. OA = osteoarthritis; HS = highly significant.\n\nThis JBS3 risk score calculator gave us three parameters about the cardiovascular condition of the heart, i.e. physiological heart age, 10-year risk in percentage of developing a CVD and life expectancy provided any other cause of death is ruled out. Statistically significant results were obtained when studying the severity of knee OA with heart age and life expectancy. Physiological heart age came out lesser but life expectancy higher for patients with a lower grade of knee OA. As we go up the K-L grade of knee OA, the physiological heart age went up and the life expectancy came down (Figure 11).\n\nThe mean physiological heart age was found to be 70 years; 10 years older than the mean chronological age (60) of the study population.\n\nThe JBS3 risk score also increased with the increase in the K-L grade of knee OA. Patients with grade 2 knee OA had a mean risk of around 11% of developing a CVD in the next 10 years and this increased with K-L grades, i.e. there was a 23% risk among the patients with grade 3 knee OA and 38% risk among those with grade 4 knee OA (Figure 12). Chi square test found this to be statistically significant {X2(4,225) = 70.776, p < .01}.\n\nThe Bonferroni post hoc analysis of K-L grade with the JBS3 risk score as the dependent variable showed multiple comparisons of each K-L grade with the others as statistically significant (Table 5). The ROC curve showing area under the curve of sensitivity vs specificity of grade 4 vs 2 and 3 for risk score is depicted in Figure 13. According to ROC analysis for the JBS3 risk score, it was determined that patients with K-L grade 4 knee OA were more likely to run a 10-year risk of developing CVD at minimum 14% with sensitivity of 72.3% and specificity of 66%. Pearson’s correlation was derived, showing a positive correlation between K-L grade and JBS3 risk score with a correlation factor of 0.57 (p < .01). Similarly, a positive correlation was seen with age, TC, SBP, BMI, and physiological heart age. A negative correlation is seen with HDL and life expectancy (Figure 14).\n\nA negative correlation was seen with HDL and life expectancy.\n\nWith JBS3 risk as dependent variable there is significant difference when all the three grades of OA knee are compared with each other. OA = osteoarthritis; HS = highly significant.\n\n\nDiscussion\n\nOsteoarthritis is a common disease in the elderly population, with its higher prevalence in the 50+ year age group well documented in the literature.1,2,3 Many studies have been done in European and American populations to measure the prevalence of OA, but there is a scarcity of studies in Asian populations. One study done by Chandra Prakash Pal et al. on the epidemiology of knee OA in India showed a prevalence of 28.7% in the study population.16 Another study in Korea showed increased prevalence with increasing age.13\n\nThe current study’s entire sample of 225 patients were above the age of 50 years. Higher prevalence of knee OA in the 50-60-year age group was seen but with no statistical significance when compared with groups of higher ages. This could be attributed to the fact that this study did not include the entire population of the given area but rather only the patients coming to orthopedic OPD with pain in the knee joint. Women are more likely to have knee pain above the age of 50 years9 and with no surprise our study also had women forming almost two thirds (60%) of the sample size. A cross-sectional study done in Korea among their population found a higher prevalence of OA in women (45%) as compared to men (25%).13 The estimated global prevalence of OA is around 10% in men and 20% in women.3,5,13,16,17 However, studies to determine the relationship between estrogen and prevalence of OA have shown contradictory results and there is no proven molecular level association.\n\nAmerican and European studies have found OA to be a disease of the upper class.17 The COPCORD study from Bangladesh also showed the same.18 On the contrary, a study conducted in south Delhi found the prevalence of OA was higher in perimenopausal women of lower classes than upper classes.19 Our study had higher numbers of patients belonging to lower and lower middle classes but showed increased severity in upper class people. Participants of lower socioeconomic status mostly had grade 2 knee OA but upper classes had the more severe grades of 3 or 4 as per K-L grading. A study in Japan revealed a higher prevalence of OA from mountainous rather than rural or urban regions, attributing the effect of activity leading to wear and tear of joints.20 Although increased activity causes more wear and tear of knee joints, there are other modifiable risk factors that also in some way directly or indirectly affect the knee joint, e.g. obesity, sedentary lifestyle, hypertension, hypercholesterolemia, smoking, diabetes mellitus and other cardiovascular risk factors.6,21,22 In the current study, BMI was found to be higher in groups with severe grades of knee OA as compared to milder grades, and there was a statistically significant association found between BMI and K-L grade of knee OA. Upper class people tend to have more modifiable risk factors,19 which is quite evident in the current study, showing increased prevalence of these modifiable risk factors (BMI, TC, HDL, SBP, DM) in these groups.\n\nOther factors like the role of systemic inflammation in the pathomechanisms of knee OA have been long studied23; however, there have been some contradictory results. Atherosclerosis, hypertension, diabetes and hypercholesterolemia cause systemic inflammation in the body, which can lead to CVD.24 Their association with knee OA has been studied by many doctors and researchers who believe that there must be some unknown relationship either directly or indirectly between CV risk factors and knee OA as they both involve systemic inflammation in the body. This belief of some sort of relationship between CV risk factors and knee OA is based on past observational studies that showed prevalence of severe grade OA in patients with CVD and vice versa. Louati K et al. in their meta-analysis of 299 publications found a high prevalence of OA in patients with DM.6 They also emphasized that metabolic OA phenotypes needs to be studied further. Huajun Wang et al.,25 in their attempt to explore the correlation between metabolic syndrome and knee OA using meta-analysis of eight studies, found a significant odds ratio (OR) even after adjusting for many risk factors. The aforementioned Korean study displayed a significantly higher knee OA prevalence in patients with hypertension and impaired glucose tolerance.13 Veronese et al. also found an association between knee OA and CVD, studying 2158 elderly participants without CVD and finding that OA at baseline was associated with subsequent incident CVD.24,26 In the current study, the mean SBP was significantly higher in patients with K-L grade 3 and 4 of knee OA. The mean SBP was 128 mmhg for K-L grade 2, 139 mmhg for K-L grade 3 and 150 mmhg for K-L grade 4. The chi square test found the differences to be statistically significant and post hoc analysis showed them to be highly significant with each increasing grade. Of the total 225 patients, 88 had a BP reading equal to or higher than 140 mmhg, most of which had a severe knee OA. Similar trends were seen while studying the prevalence of DM in these patients. In in the total sample, 25.8% patients had DM, yet the largest percentage (53%) was seen in patients with K-L grade 4 knee OA, showing the risk of DM increases with severity of knee OA, as found by Louati K et al.6 Hypercholesterolemia was also studied here, being another systemic inflammatory marker. It showed a statistically significant relationship with K-L grades of knee OA; mean serum TC was found to be higher in patients with severe grades of knee OA, whilst mean serum HDL was found to be lower in patients with severe grades of OA.\n\nSystolic BP, DM and hypercholesterolemia are three of the major contributors to CVD.13 As per the results of this study it would be safe to say that patients with severe knee OA have a higher risk of developing a CVD in the future. On the contrary, Hoeven et al.27 believe that OA-related disability and not OA predicts CVD. They postulated that neither clinical nor radiological knee OA predicts a CV event in the future, rather it’s the disability, independent of OA, that predicts the OA. This is along the lines of belief of many other researchers who postulate that OA doesn’t directly lead to a CVD, rather it renders a patient physically more inactive and disabled than those without OA, which ultimately increases the CV risk factors. Whether OA causes CVD directly or indirectly, it is hard to ignore the fact that they often exist co-dependently in a patient’s body. Assessment of a patient with knee OA for CV risk factors would be of no harm and rather provide the doctor a systemic approach to the patient’s morbid condition and also make the patient aware about their unknown CV risk factors and risk of developing a CVD. Assessment of CV risk factors in patients with OA has been a topic of research in Korea. Jeong et al. studied data from the Korean National Health Survey and Nutrition Examination Survey (KNHANES) and found a higher prevalence of hypertension, DM, dyslipidemia, angina and myocardial infarction in patients with OA compared with healthy individuals.28\n\nContrary to our assumption, smoking was not found to have any statistically significant effect on the K-L grade of knee OA. However, it has a role to play in determining the increased risk of CVD in these patients.29 Other variables such as atrial fibrillation, chronic kidney disease and rheumatoid arthritis were not found to be prevalent in our study population, preventing us from analyzing any relationship between them and knee OA.\n\nThis study has attempted to collaborate the basic mechanical, causal and shared risk factors (age, obesity, gender) between knee OA and CVD. It has also tried to study various other CV risk factors (hypertension, DM, dyslipidemia, smoking, family history) in patients with OA. Ho Sun Kim et al.13 used the Framingham risk score (FRS) in south Koreans to study the association between OA and CV risk factors; however, Manish Bansal et al.30 found that the FRS cannot be applied efficiently to the Indian population. They did, however, find the JBS3 risk score calculator to be the best predictor of a CV event in high risk Indians. We used the JBS3 risk score to calculate 10-year risk of developing CVD, physiological heart age and life expectancy. Statistically significant data revealed the physiological heart age of the patients to be much higher than their actual age and their life expectancy shortened with more CV risk factors. The shortened life expectancy was much more common in patients with severe OA as they also had a higher JBS3 risk of developing CVD over the next 10 years. It was observed that the mean physiological heart age was as much as 10 years higher than the mean age for the entire study population, drawing a strong correlation between OA and the cardiovascular system.\n\nThe mean JBS3 risk was 11% for patients with grade 2 OA, 23% for patients with grade 3 OA and 38% with grade 4 OA. This was along the same lines as studied by Ho Sun Kim et al. in Korea,12 where multiple comparisons were made within the different grades of knee OA while studying the relationship with JBS3 risk and all were found to be highly significant. Our study had 225 patients with a prevalence of grade 4 knee OA of 13%. With the help of the ROC curve, the cut-off value of JBS3 was calculated to be 14%, which implies the amount of risk patients with grade 4 knee OA had of developing a CVD in the next 10 years, with a sensitivity of 72.3% and specificity of 66%. It would not be an overstatement to say that a patient with grade 4 knee OA undergoing total knee replacement could have a CV risk of as much as 14%.\n\nThis is the first study of its type to be conducted in this part of the world. India, although one of the countries where both OA and CVD is most prevalent, has very little literature stating the relationship between the two. The strength of this study lies in the different variables (demographic and CV risk factors) that were studied. With the genetic and phenotypic variations in the population in this part of the world, BMI plays a major role in understanding various pathologies in human body. The BG Prasad scale has helped us to divide the study population into various socio-economic strata and study its relation to both OA and CV risk factors. In addition, the diagnosis of knee OA has been made with the help of standard radiographs using K-L classification. The potential confounding factors (CV risk factors) are clubbed together with the help of a standardized risk score calculator devised by the Joint British Society. JBS3 also tells us about the physiological heart age and life expectancy, which may be easier for patients to comprehend. Although these factors have an individual effect on the pathomechanisms of knee OA, when they are used as a part of this scale, various limitations are eliminated, and they are studied as a whole. The risk factors thus are studied both individually as well as co-dependently. However, this study has its limitations too. It is a cross-sectional study, which comes with its own inherent limitations. The study population only has patients with K-L grade of ≥2, so the risk assessment of patients with K-L grade 1 was not done here. The JBS3 risk score calculator used here could only come close to being ideal for the study population of our country. A risk score calculator developed in our country for Indian genotypes and phenotypes would be ideal. Also, it doesn’t include stress as a variable, which has a known effect on cardiovascular status.31 The SBP values entered here are of single reading, which can be affected by the hemodynamic status of the patient at one particular moment. The readings of TC and HDL values may have differed depending on the investigator and equipment available from place to place. Some variables of this tool depend upon the history given by the patient, which has its own limitations. The JBS3 risk score calculator is not a tool to initiate treatment for a patient, rather a tool that gives a fair idea of the patient’s general condition and CV status.\n\n\nConclusion\n\nOur study concluded that there is a strong positive correlation between knee OA and CVD with CV risk score being directly proportional to the severity of OA. JBS3 is a comprehensive risk score calculator as well as a screening tool that produces three more variables, namely 10-year risk of developing CVD, physiological heart age and life expectancy, which are easy to comprehend.\n\nThe JBS3 risk score calculator, a comprehensive tool, can be applied to the general population in routine clinical practice to comprehensively address the CV risk factors in patients with knee OA. At the same time, screening for the same risk factors can be carried out in unaware high-risk patients. A population-based study with similar tools can help us derive a JBS3 risk score cut-off, which would aid in management of patients undergoing total knee replacement. The JBS3 risk score calculator/mobile-based application could help health care professionals to better illustrate the risk of CVD and the gains that can be made by early interventions.\n\n\nData availability\n\nDryad: Assessment of cardiovascular risk factors in patients with Knee Osteoarthritis. https://doi.org/10.5061/dryad.79cnp5htv.32\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\nBedson J, Jordan K, Croft P: The prevalence and history of knee osteoarthritis in general practice: a case–control study. Fam Pract. 2005; 22: 103–8. Publisher Full Text\n\nDillon CF, Rasch EK, Gu Q, et al.: Prevalence of knee osteoarthritis in the United States: arthritis data from the Third National Health and Nutrition Examination Survey 1991–1994. J Rheumatol. 2006; 33: 2271–9. PubMed Abstract\n\nKellgren JH: Osteoarthrosis in patients and populations. Br Med J. 1961; 2: 1–6. 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Indian Heart J. 2014; 66(6): 580–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSumner JA, Khodneva Y, Muntner P, et al.: Effects of Concurrent Depressive Symptoms and Perceived Stress on Cardiovascular Risk in Low- and High-Income Participants: Findings From the Reasons for Geographical and Racial Differences in Stroke (REGARDS) Study. J Am Heart Assoc. 2016 Oct 10; 5(10): e003930. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoel S: Assessment of cardiovascular risk factors in patients with Knee Osteoarthritis. Dryad, Dataset. 2021. Publisher Full Text" }	[ { "id": "92755", "date": "09 Sep 2021", "name": "Win Min Oo", "expertise": [ "Reviewer Expertise Osteoarthritis", "Musculoskeletal imaging", "Neurorehabilitation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt’s been a great pleasure reviewing this manuscript. The authors examined various cardiovascular (CV) risk factors in 225 patients with knee OA, concluding that a strong positive correlation between knee OA and CVD existed.\nGenerally, the manuscript is well written though there is still a large space for much improvement. I have several comments to improve it.\nAbstract:\nIn the conclusion section, we would suggest omitting some sentences as it is not appropriate to mention them here. Perhaps the best place would be in the methodology as the outcome measure: \"JBS3 is a comprehensive risk score calculator as well as a screening tool, which…\".\nIntroduction:\nThird paragraph: Generally, systemic inflammation is not part of the OA disease process nor florid as in rheumatoid arthritis which has an obvious association with CVD.\n\nReferences 3 and 4 you used for OA being associated with high cholesterol levels did not mention such - page 3, third paragraph: ”recent studies have reported that OA is associated with high serum cholesterol levels in females”.\nMethodology: Study design\nDo you mean that a convenience sample was used? Any retrospective data? If so, please mention it.\nStudy participants\nJust wondering why the American College of Rheumatology criteria was not used for case definition. How are references 9 and 10 appropriate here, especially for case definition?\n\nFor the sample size calculation, the estimated proportion was taken from a Korean study in which the prevalence of knee OA may be different from an Indian population, and it may be overestimated as the study was not conducted in the community setting.\n\nAre there any Indian studies for the record of community prevalence of knee OA? We think it would be more relevant if available.\nVariables\nIn knee X-rays, did you consider only patellofemoral or tibiofemoral OA or both eligible?\n\nPhysical inactivity (sedentary lifestyle) and NSAID usage should be included and adjusted as the potential confounders.\n\nStatistical analysis\nJust wondering why the multiple regression model for adjusting the confounders was not used.\n\nFor post-hoc analysis, did you conduct any correction of the p-value for multiple comparisons? If so, please mention it here.\nResults\nInstead of using many figures for each of the baseline characteristics, one table should work well for all variables. Strongly suggest doing so.\n\nTable 2 is difficult to understand without the inputs of blood pressure calculated. Suggested to modify it.\n\nToo many figures and interpretations for a research article. Please be concise.\n\nThe current or previous medication used for OA should be reported as NSAID especially Cox-2 inhibitors may increase the CVD risk and morbidity. Therefore, such confounders should be adjusted in the statistical analysis to draw a valid interpretation.\nDiscussion\nToo long and involves the repetition of much of the results section instead of correlating with the findings of other papers.\n\nThe redundancy of the language should be avoided, i.e. \"This is the first study of its type to be conducted in this part of the world. India...\".\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": "7390", "date": "25 Nov 2021", "name": "Sagar Goel", "role": "Author Response", "response": "Thank you for your feedback. We have tried our best to respond to some of your queries and made some modifications to the manuscript as per your suggestions. Abstract: In the conclusion section, we would suggest omitting some sentences as it is not appropriate to mention them here. Perhaps the best place would be in the methodology as the outcome measure: \"JBS3 is a comprehensive risk score calculator as well as a screening tool, which…\". Author response: Done. Introduction: Third paragraph: Generally, systemic inflammation is not part of the OA disease process nor florid as in rheumatoid arthritis which has an obvious association with CVD. Author response: Done. References 3 and 4 you used for OA being associated with high cholesterol levels did not mention such - page 3, third paragraph: ”recent studies have reported that OA is associated with high serum cholesterol levels in females”. Author response: The text has been modified and the concerned reference is added. Methodology: Study design Do you mean that a convenience sample was used? Any retrospective data? If so, please mention it. Author response: Yes, a convenience sample was used. Moreover, retrospective data was also collected as mentioned under the heading Ethics Statement and Sampling: “Also, case records of patients diagnosed with knee OA between January 2017 to September 2018 were studied…” Study participants Just wondering why the American College of Rheumatology criteria was not used for case definition. How are references 9 and 10 appropriate here, especially for case definition? Author response: We believed Kellgren and Lawrence grading of OA knee based on the X-ray was more suitable for case definition here. Using reference no 9, we decided to keep 50 years of age as a cutoff for patient selection. For the sample size calculation, the estimated proportion was taken from a Korean study in which the prevalence of knee OA may be different from an Indian population, and it may be overestimated as the study was not conducted in the community setting. Are there any Indian studies for the record of community prevalence of knee OA? We think it would be more relevant if available. Author response: The Prevalence of knee OA among Indian and Korean Populations is comparable and has been mentioned with reference Variables In knee X-rays, did you consider only patellofemoral or tibiofemoral OA or both eligible? Author response: Both were considered. Physical inactivity (sedentary lifestyle) and NSAID usage should be included and adjusted as the potential confounders. Author response: We used the JBS3 risk score calculator which did not include physical inactivity and NSAID usage as variables. Statistical analysis Just wondering why the multiple regression model for adjusting the confounders was not used. Author response: Done and added. For post-hoc analysis, did you conduct any correction of the p-value for multiple comparisons? If so, please mention it here. Author response: Yes, it was done. Results Instead of using many figures for each of the baseline characteristics, one table should work well for all variables. Strongly suggest doing so. Author response: Done. Table 2 is difficult to understand without the inputs of blood pressure calculated. Suggested to modify it. Author response: Done. Too many figures and interpretations for a research article. Please be concise. Author response: Done. The current or previous medication used for OA should be reported as NSAID especially Cox-2 inhibitors may increase the CVD risk and morbidity. Therefore, such confounders should be adjusted in the statistical analysis to draw a valid interpretation. Author response: We used the JBS3 risk score calculator which did not include physical inactivity and NSAID usage as variables. Discussion Too long and involves the repetition of much of the results section instead of correlating with the findings of other papers. Author response: Done. The redundancy of the language should be avoided, i.e. \"This is the first study of its type to be conducted in this part of the world. India...\". Author response: Done." } ] } ]	1	https://f1000research.com/articles/10-508
https://f1000research.com/articles/11-204/v1	17 Feb 22	{ "type": "Research Article", "title": "Assessment of patient satisfaction toward pharmaceutical benefit package provided by a health insurance corporation of Khartoum State", "authors": [ "Ahmed Osman Mohamed", "Ahmed Shoaib Hussain", "Manasik Omar Alhaj", "Ahmed Shoaib Hussain", "Manasik Omar Alhaj" ], "abstract": "Background: Patient satisfaction is an important measure of health service and a key indicator of the quality of health service. Studies focus on how to improve quality rather than patient satisfaction. This study was conducted to identify patients’ satisfaction with the pharmaceutical service package of Health Insurance Corporation of Khartoum State in Jabal-Awliya locality, Khartoum, Sudan. Methods: A cross-sectional study was conducted between January and August 2020. Data were collected using a structured questionnaire. Satisfaction was estimated using the Likert Scale. The association between patient’s satisfaction and patient care indicators, namely: average dispensing time, percentage of medicine dispensed and labelled, and patient correct dose knowledge were assessed using Chi- square test, where a p-value < 0.05 was considered significant.\n\nResults: Out of 378 participants, the mean age was 47 with a comparable frequency of male and female participants (49.3% and 50.7% respectively). The mean satisfaction was 62.2% (3.11 ± 0.68). Most of the participants were satisfied with the way the pharmacist dealt with them (3.7, ± 0.778), while a low mean of satisfaction was reported regarding the availability of medicine within the pharmaceutical benefit package (2.06, ± 1.17). Average dispensing time was 5.78 minutes (p = 0.002), a low percentage of medicine actually dispensed and labelled was observed at 67% and 58% respectively (p = 0.00). A higher patient knowledge was reported 96.6% (p = 0.00), and the majority of the patients were able to pay 65% (p = 0.00). Conclusion: The current study demonstrates a comparable satisfaction score. However, medication unavailability is the main factor that affects patient satisfaction.", "keywords": [ "HICKs", "Patient satisfaction", "Pharmaceutical benefit package", "Sudan." ], "content": "Introduction\n\nFollowing the dramatic change in the role of pharmacists observed in the last decades to be more patient centred, patient satisfaction has emerged as a prerequisite in pharmaceutical care. It can be defined as personal appraisal of the pharmaceutical care; it is a subjective and multidimensional measure which answers the questions about what patients expect and what patients find.1,2\n\nAchieving satisfaction is the best way to convince the patient about the health services provided, ensuring adherence to therapy and consequently improving health outcome. Therefore, patient satisfaction has driven the attention of developed as well as developing countries; in fact there have been several attempts to develop a validated tool to measure the patient satisfaction.2–5\n\nPatient care indicators (PCIs) are one of World Health Organization's (WHO) core drug use indicators, they are used to reflect the quality of the pharmaceutical services delivered by the health facility by describing what the patient experienced at the health facility and to what extent the patient has been trained to deal with the pharmaceuticals that have been dispensed, through measuring several factors including: average consultation time (ACT), average dispensing time (ADT), percentage of drugs actually dispensed (PMAD), percentage of drugs actually labelled (PMAL), and patients’ knowledge of correct dosage (PKCD). Given their vital role in determining the quality of the pharmaceutical service, the WHO have comprehensively described PCIs in terms of providing detailed protocol of how to measure each parameter as well as setting out the optimal range of each indicator.6,7\n\nPrevious studies from Egypt, Nigeria, Kingdom of Saudi Arabia (KSA), Ethiopia, Pakistan, and Spain have provided detailed descriptions of one or all PCIs in either a hospital or out-patient pharmacy.6,8–11 A similar study was conducted recently in Sudan, where the pattern of drug use by using WHO's PCIs was estimated in four hospitals in Khartoum state.7\n\nHealth Insurance Corporation of Khartoum State (HICKS) is one of leading Social Health Insurance (SHI) organizations limited to Khartoum State which provides services to primary healthcare facilities of the State Ministry of Health and the Federal Ministry of Health hospitals. The HICKS receives its funds mainly through the State Ministry of Health and house-holds (36.5% and 27% respectively).12 Other sources of funding are parastatal organizations and private employers’ contributions. Despite the presence of SHI in Sudan for more than 20 years, universal coverage has not been achieved yet; it faces many obstacles such as rising healthcare costs due to inflation and lack of affordability by beneficiaries. Moreover, the quality of the services provided including patient satisfaction is poorly understood.12 This study was conducted to measure the overall patient satisfaction towards the Pharmaceutical Benefit Package provided by HICKS and to predict the possible association between PCIs and overall satisfaction.\n\n\nMethods\n\nA cross-sectional study based on STROBE statement was conducted in Jabal-Awliya locality in Khartoum state, Sudan, in the period from January to August 2020.\n\nThe study samples were outpatients seeking pharmaceutical care under the umbrella of HICKS. All adults registered at HICKS aged over 18 were asked to participate in the study when visiting one of the HICKS pharmacies.\n\nThe HICKS reported that the average monthly frequency of patients attending to their pharmacy at Jabal-Awliya locality (47 pharmacies) was 22084 (data was kindly provided by HICKS, Research & Development Department). By using Solvin's equation,13 the sample size (378) was calculated using the known frequency as follows:\n\nWhere;\n\nn = Sample size, N = Targeted population attending at different pharmacy, e = Margin of error (0.05) at 95% confidence level.\n\nA semi-structured questionnaire was administered to the study participants for data collection which was adopted from the previous studies.1,14 At the beginning of the questionnaire, statement for consent was included (verbal consent was considered in case of participants who were illiterate) to allow the use of their data. The questionnaire was divided into three main parts. Part one includes demographic data (age, gender, and visiting time). Part two includes queries about satisfaction, with 10 closed questions based on a 5-point Likert scale on disagreement/agreement15: strongly disagree 1, disagree 2, not sure 3, agree 4, and strongly agree 5. Based on these points (from 1-5), satisfaction was calculated by taking the mean of the 10 questions (part one and two were filled by participants). Part three includes questions about factors that might potentially affect satisfaction according to WHO standard measurement of PCIs,16 which include ADT (time taken for dispensing in minutes), PMAD (percentage of medicine dispensed and medicine in the prescription), PMAL (percentage of medicine labeled and medicine dispensed), and evaluation of patient correct dose (did the patient know the correct dose or not). An additional question regarding patients’ ability to pay despite their involvement in HICKS was also included (was the patient able to pay or not). The third part of the questionnaire was filled by the investigators through face-to-face interviews of the participants after completing parts one and two. The data was collected by three investigators. The questionnaire was originally written in English and translated by a language expert to the local language (Arabic) and translated back to English to ensure consistency. A pilot study was conducted (15 participants) prior to gross data collection for the purposes of examining the validity of the questionnaire, the results of which were excluded in the study analysis. Relevant modifications were addressed.\n\nThe data were coded, entered, and analyzed using IBM® SPSS Statistic, Version 19 for Windows. Descriptive statistics were conducted using frequency and proportions. Chi-square was used to determine the relationship between different variables. P-value of 0.05 or less was considered statistically significant.\n\nEthical approval was obtained from Ahfad University for Women Research Ethics Committee and HICKS prior data collection (approval date 15 January 2020). Verbal informed consent was collected from participants who were illiterate, and written informed consent obtained from the rest of the participants. Personal identities were not recorded throughout the studies to ensure confidentiality.\n\n\nResults\n\nA total of 378 participants were included in the study; there was no missing data as investigators were able to detect and resolve this while the questionnaires were being completed. The mean age was 47.23, ranging from 18 to 90 years old. The frequency of each gender and the percentages are shown in Table 1. Most of the included participants visit the selected pharmacies in the morning (70%), while others visit the pharmacy at evening shift.\n\nRegarding the patients’ satisfaction, the overall mean of satisfaction was 3.11 ± 0.68 (62.2%, calculated by dividing the sum of each participant satisfaction score over total number of participants); the minimum reported satisfaction was 1.40 while the maximum was 4.50. Complete questions that were used to measure satisfaction along with their statistics expressed in mean and standard deviation are shown in Table 2.\n\nThe WHO PCIs\n\nWith respect to the PCIs recommended by WHO which are related to the pharmaceutical care: 40% of the participants have, on average, less than 5 minutes dispensing time, while 60% have an average of 5-25 minutes dispensing time (Table 3). Regarding the availability of medicine: more than half (55%) of the patients find less than 75% of their prescribed medicine. Regarding the percentage of medicine labelled: one out of four (24.6%) have less than 25% of their medications labelled properly and 42.6% have more than 75% their medications labelled properly (Table 3). 96.7% of the participants have a good knowledge of the correct dose and as few as 3% do not have enough knowledge (Table 3). Regarding the ability of the patients to pay despite the health insurance: nearly two out of three (64.7%) are able to pay (Table 3).\n\nAs shown in Table 4, by using Chi-square test the analysis reveals that there was no statistically significant association between satisfactions and gender or visiting time (P value = 0.92 and 0.88 respectively).\n\n** Significant P-value.\n\nA significant association between the five included parameters of PCIs and satisfaction was observed in the analysis using Chi-square test.\n\nRegarding the average dispensing time (ADT), participants with more than 5 minutes ADT were found to be more satisfied (P = 0.002). With respect to the percentage of medicine dispensed and labelled: those participants with a percentage of medicine either dispensed or labelled higher than 75% were more satisfied (P = 0.00). Moreover, patients with knowledge of the correct dose and ability to pay were found to be more satisfied with a statistically significant P-value (0.00). Association between PCIs and mean of satisfactions are shown in Table 4.\n\n\nDiscussion\n\nPatient satisfaction is one of the most important factors that affects medication adherence and subsequently treatment success/failure. In this study, a total of 378 questionnaires were distributed to out-patients who sought their medication at one of the HICKS pharmacies with the aim of assessing the level of satisfaction. The overall satisfaction was 62.2% and all PCIs were found to be associated with patient satisfaction.\n\nThe mean age of the participates was 47 years old, with 49.3% male and 50.7% female participants (Table 1). A study conducted in Canada aiming to validate published satisfaction scales in larger and more diversified patient populations included 682 patients and reported that the percentages of male and female participants were 36.9% and 63.1% respectively.17 In another study setting in Peru, a slightly lower mean of age (42.8) was reported, while the percentage of the females was 59.6% and males was 40.4%.18 A similar study that was conducted in United Arab Emirates (UAE) reported a lower mean of age across the participants of 34, and in the same study, the percentages of males and females was 39% to 61% respectively.3 This slight fluctuations in age and gender among the studies might be due to the difference in study population, area and sample size.\n\nIn this study, the mean of satisfaction was found to be 3.11 (62.2%). This finding is comparable to the finding in a Nigerian study (60.4%),19 and higher than the result obtained in Ethiopia and Pakistan in 2019 and 2018 respectively,8,9 in which the satisfaction was reported to be 51% and 55.6% respectively. On the other hand, this level of satisfaction is lower than that obtained in Spain and South Korea (76% and 74.6% respectively).2,20 This variation is probably due to the fact that each study adopts different tools to assess satisfaction and undoubtedly reflects the difference in the services in the different countries.\n\nThe current study reveals that the participants were highly satisfied with the pharmacist who provided the health services; an over 70% satisfaction rate was reported by the participants to the way the pharmacists are dealing with them, care to inquiries and clarity of instructions (Table 2). Similar to this figure; Spanish, Pakistani and Ethiopian participants were also satisfied with the approach or communication of the health professional providing the services.8,9,20 Fortunately, knowledge and practice of the pharmacy staff are the most important factors for delivering a better pharmaceutical service and hence improving patient satisfaction. In contrast, the study reveals the weakest domain which affects patient satisfaction: unavailability of all medications under the umbrella of health insurance (41.2%) was the least rated factor (Table 4). A higher figure was obtained by several studies conducted in Pakistan, UAE and in some regions in Ethiopia with 63.8%, 70% and 59.6% respectively,3,8,9 however, a study conducted in different regions in Ethiopia reported a lower percentage 33.1%.21 In this context, while the study reports a relatively higher overall satisfaction (62.2%) this finding is alarming since several studies demonstrate a significant association between satisfaction and medication availability; this is considered a hidden factor that might negatively impact the whole medical care.\n\nIn this study, parameters that are used to measure rational use of medicine which are associated with patient satisfaction were considered. The ADT was 5.78 minutes in which 60% of the participants have more than 5 minutes ADT. A significant association (P = 0.002) between ADT and overall satisfaction was reported in this analysis (Tables 3 and 4). The reported ADT is two times higher than that reported in India (3 minutes) and much higher than that reported in KSA (1.5 minutes) and Kuwait (1 minute).6,22,23 It is known that short ADT is insufficient to explain the dosage regimen, adverse effects of drugs and all precautions, while the reported ADT is longer in comparison to the previous studies, this might be due to the fact that the pharmacist spent a longer time explaining to the patient the insured and non-insured medication in the prescription as HICKS do not cover full insurance for all medication.\n\nThe percentage of medicine dispensed was 67%; this figure was lower than the WHO recommended percentages (100%). The obtained PMD was statistically associated with satisfaction (P = 0.00) (Table 4). It is lower than that reported in Egypt, Kenya and KSA,6,24,25 and slightly higher than a previous study conducted in Sudan (58%).7 This finding reflects the huge medicine shortage in the health system, where adequate supply is the corner stone in the health system and if left unmanaged, health crises might arise.\n\nThe WHO recommended that the proper labelling must include patient name, dose regimen and drug use. Due to the unavailability of proper labelling machines, labelling was considered proper if the dose regime and drug use were hand written correctly. In this regard, the PML was 58% (Table 3), a far lower percentage than WHO recommendation (100%), and even lower than that reported in Kenya, Kuwait, and KSA.6,23,25 This finding indicates the poor labelling practise among the pharmacists which might be due to lack of knowledge or because some patients object to the labelling since they take these medications on a regular basis.\n\nRegarding patient knowledge, a slightly lower percentage (96.6%) than the WHO recommendation (100% knowledge) was obtained (Table 3); similar figures were reported in Kenya (96%) and Egypt (94%),24,25 while it is higher than that reported previously in Sudan and KSA (79%).6,7 This could be attributed to fact that most of the coverage medications were for chronic diseases taken regularly by the patients. With respect to patient's ability to pay despite health insurance, one out of three patients are not able to pay (Table 3). This is an alarming finding, as it is known that ability to pay is affected by out-of-pocket costs (expenses for medical care that aren't reimbursed by insurance) which is considered to be a significant barrier to accessing or maintaining care for those with chronic diseases,26 and consequently might make the whole health service valueless.\n\nOne of the key limitations in this study is the study area: since it was conducted in one locality in Khartoum, a more diverse area with a larger sample could provide a more robust result. Moreover, this study used interview questionnaires which were conducted at the time of patients visiting the pharmacy and hence it is subjected to social desirability bias.\n\n\nConclusions\n\nThe overall satisfaction score was (62%). However, medication unavailability remains to be a hidden factor which undoubtedly affects the patient satisfaction.\n\n\nData availability\n\nfigshare: Assessment of Patient Satisfaction towards Pharmaceutical Benefit Package Provided by Health Insurance Corporation of Khartoum State. https://doi.org/10.6084/m9.figshare.18129206.v4.27\n\nThis project contains the following files:\n\nResearch Data.xlsx (complete data set for each participant response).\n\nfigshare: Questionnaire used data collection. https://doi.org/10.6084/m9.figshare.19107269.v1.28\n\nThis project contains the following files:\n\nSatisfaction survey.pdf (the questionnaire used for data collection).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nAuthors greatly appreciate the assistance of Research & Development Department at HICKS for helping with sampling.\n\n\nReferences\n\nLarson LN, Rovers JP, MacKeigan LD: Patient satisfaction with pharmaceutical care: update of a validated instrument. J. Am. Pharm. Assoc. (1996). 2002; 42: 44–50. PubMed Abstract \| Publisher Full Text\n\nLee S, Godwin OP, Kim K, et al.: Predictive factors of patient satisfaction with pharmacy services in South Korea: A cross-sectional study of national level data. PLoS One. 2015; 10: e0142269. PubMed Abstract \| Publisher Full Text\n\nHasan S, Sulieman H, Stewart K, et al.: Assessing patient satisfaction with community pharmacy in the UAE using a newly-validated tool. Res. Soc. Adm. Pharm. 2013; 9: 841–850. PubMed Abstract \| Publisher Full Text\n\nKraska RA, Weigand M, Geraedts M: Associations between hospital characteristics and patient satisfaction in Germany. Health Expect. 2017; 20: 593–600. PubMed Abstract \| Publisher Full Text\n\nStepurko T, Pavlova M, Groot W: Overall satisfaction of health care users with the quality of and access to health care services: a cross-sectional study in six Central and Eastern European countries. BMC Health Serv. Res. 2016; 16: 1–13. Publisher Full Text\n\nEl Mahalli A, Akl O, Al Dawood S, et al.: Salem A (2012) WHO/INRUD patient care and facility-specific drug use indicators at primary health care centres in Eastern province, Saudi Arabia. EMHJ-Eastern Mediterranean Health Journal. 2012; 18(11): 1086–1090. PubMed Abstract \| Publisher Full Text\n\nTm H, Saeed A, Awad M: Evaluation of Drug Use at Four Hospitals in Khartoum, Sudan. Int. J. Qual. Health Care. 2020; 3: 2642.\n\nSemegn S, Alemkere G: Assessment of client satisfaction with pharmacist services at outpatient pharmacy of Tikur Anbessa Specialized Hospital. PLoS One. 2019; 14: e0224400. PubMed Abstract \| Publisher Full Text\n\nAziz MM, Ji W, Masood I, et al.: Patient satisfaction with community pharmacies services: A cross-sectional survey from Punjab; Pakistan. Int. J. Environ. Res. Public Health. 2018; 15: 2914. PubMed Abstract \| Publisher Full Text\n\nAfolabi MO, Erhun WO: Patients\\'response to waiting time in an out-patient pharmacy in Nigeria. 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Simply Psychology. 2019 August 3; 2019.\n\nOrganization WH: Promoting rational use of medicines: core components. World Health Organization; 2002.\n\nKassam R, Collins JB, Berkowitz J: Patient satisfaction with pharmaceutical care delivery in community pharmacies. Patient Prefer. Adherence. 2012; 6: 337. Publisher Full Text\n\nAlarcon-Ruiz CA, Heredia P, Taype-Rondan A: Association of waiting and consultation time with patient satisfaction: secondary-data analysis of a national survey in Peruvian ambulatory care facilities. BMC Health Serv. Res. 2019; 19: 439. PubMed Abstract \| Publisher Full Text\n\nOparah AC, Enato EF, Akoria OA: Assessment of patient satisfaction with pharmaceutical services in a Nigerian teaching hospital. Int. J. Pharm. Pract. 2004; 12: 7–12.\n\nMárquez-Peiró JF, Pérez-Peiró C: Evaluation of patient satisfaction in outpatient pharmacy. Farmacia Hospitalaria (English Edition). 2008; 32: 71–76. Publisher Full Text\n\nDesalegn AA: Assessment of drug use pattern using WHO prescribing indicators at Hawassa University teaching and referral hospital, south Ethiopia: a cross-sectional study. BMC Health Serv. Res. 2013; 13: 1–6. Publisher Full Text\n\nSingh S, Dadhich A, Agarwal A: A study of prescribing practices in a tertiary care hospital using WHO core indicators. Asia pacific journal of pharmacology. 2003; 16: 9–14.\n\nAtif M, Sarwar MR, Azeem M, et al.: Assessment of core drug use indicators using WHO/INRUD methodology at primary healthcare centers in Bahawalpur, Pakistan. BMC Health Serv. Res. 2016; 16: 684. PubMed Abstract \| Publisher Full Text\n\nAkl OA, El Mahalli AA, Elkahky AA, et al.: WHO/INRUD drug use indicators at primary healthcare centers in Alexandria, Egypt. J. Taibah Univ. Med. Sci. 2014; 9: 54–64. Publisher Full Text\n\nNyabuti AO, Okalebo FA, Guantai E: Examination of WHO/INRUD Core Drug Use Indicators at Public Primary Healthcare Centers in Kisii County, Kenya. medRxiv. 2020.\n\nDodd R, Palagyi A, Guild L, et al.: The impact of out-of-pocket costs on treatment commencement and adherence in chronic kidney disease: a systematic review. Health Policy Plan. 2018; 33: 1047–1054. PubMed Abstract \| Publisher Full Text\n\nKunna A: Assessment of Patient Satisfaction toward Pharmaceutical Benefit Package Provided by Health Insurance Corporation of Khartoum State. figshare. Dataset. 2022. Publisher Full Text\n\nKunna A: Questionnaire used data collection. figshare. Dataset. 2022. Publisher Full Text" }	[ { "id": "124106", "date": "07 Mar 2022", "name": "Masoud Mohammadnezhad", "expertise": [ "Reviewer Expertise Public Health and Health Promotion" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good study that applied a proper study design. There are a few comments that I advise authors to address at this stage:\nPlease write where the study is exactly conducted? Why this study setting is chosen?\n\nWhat were the exclusion criteria?\n\nIt is better to consider 5-10% as a non-respondent rate when you calculated the sample size.\n\nWhat was the sampling method?\n\nPlease write what will be the lowest and highest score for the questions used in part two.\n\nPlease write \"average dispensing time\" where you wrote ADT.\n\nIt seems you used both structured and semi-structured questionnaires.\n\nWhy did you use three investigators to collect data? As the third part of the questionnaire was a semi-structured questionnaire, it was better to useone investigator to have the same record of participants' answers.\n\nHow did you use the results of the pilot study in validation of the questionnaire? Did you make any changes to the questionnaire?\n\nDid you exclude 15 people who participated in the pilot study from the main study?\n\nIn the result section and its last paragraph, please just use ADT instead of writing its complete words.\n\nPlease expand the conclusion section and try to provide some recommendations based on the findings of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "175143", "date": "04 Jul 2023", "name": "Mostafa A S Ali", "expertise": [ "Reviewer Expertise Drug Safety", "Hospital Pharmacy services", "Therapeutic Drug Monitoring Pharmacovigilance Clinical Studies", "Patient Safety", "Pharmacoepidemiology", "Pharmaceutical Education and Medication Adherence" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 used a questionnaire to measure patient satisfaction with pharmacy services which is an important part to ensure the quality of healthcare services.\n\nI have some inquiries from the authors:\n\nThe rationale and objective of conducting the study need to be expanded and clarified at the end of the interdiction section\n\nThe authors stated in the Method section that they adopted the questionnaire from the previous studies references 1 1 and 14 2. However, reference 1 contains 20 items questionnaire and reference 14 is a WHO report on the role of the pharmacist in the healthcare system. Therefore, the internal consistency and validation of the new scale structure of the selected items needs to be assessed. There is no mention about the validation and reliability from the pilot study.\n\nIn the Result section whenever, you report mean age or mean value it better to insert the Standard Deviation.\n\nTable 1 missed some demographics such as the education level and type of patient visit to the pharmacy (first visit or frequent visit) because it may affect the satisfaction rate\n\nIn the association between the overall satisfactions and demographic factors, how you assessed or described the overall satisfaction in Table 4? was it a binary variable poor satisfied or highly satisfied (what is the cut-off score?), or your outcome was 5 point Likert score from poor to high scores? I think you need to define the outcome.\n\nIn paragraph 2 of the Discussion section, what the point of just comparing the different proportions of male and female participants between your study and other studies if it did not make gender -associated difference in satisfaction rate? I find it useless.\n\nIn paragraph 3 of the Discussion section, the authors compared the overall satisfaction rate with other countries, what about the satisfaction rate previously reported in Sudanese studies?\n\nThe conclusion need to be expanded to include more factors affecting patient satisfaction such as average waiting time.\n\nSome sentences have excess words such as \"In this study, the mean of satisfaction was found to be 3.11\" that could be simple like \"In this study, the mean of satisfaction was 3.11\". there are other similar sentences\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/11-204
https://f1000research.com/articles/11-202/v1	16 Feb 22	{ "type": "Systematic Review", "title": "Clinical outcomes of COVID-19 patients with solid and hematological cancer: a meta-analysis and systematic review", "authors": [ "Joni Wahyuhadi", "Fadhillah Putri Rusdi", "I G. M. Aswin R. Ranuh", "Rizki Meizikri", "Irwan Barlian Immadoel Haq", "Rahadian Indarto Susilo", "Makhyan Jibril Al Farabi", "Fadhillah Putri Rusdi", "I G. M. Aswin R. Ranuh", "Rizki Meizikri", "Irwan Barlian Immadoel Haq", "Rahadian Indarto Susilo", "Makhyan Jibril Al Farabi" ], "abstract": "Background: Previous research has consistently shown the significant difference in outcome between cancerous and non-cancerous patients with coronavirus disease 2019 (COVID-19). However, no studies have compared the clinical manifestation of COVID-19 in hematologic cancers patients and solid cancers patients. Therefore, we analyzed the outcome of COVID-19 patients with hematological cancer and primary solid cancer worldwide through a meta-analysis and systematic review. Methods: This meta-analysis and systematic review included English language articles published between December 2019 – January 2021 from Pubmed and Google Scholar. The Newcastle Ottawa Score was used to assess the quality and bias of included studies. The outcome measures were case-fatality rate and critical care events for COVID-19 patients with cancer and comorbidities. Results: The initial search found 8910 articles, of 20 were included in the analysis. Critical care events and mortality were higher in the hematological than primary solid cancer group (relative risk (RR)=1.22 & 1.65; p <0.001). Conversely, mortality was lower in patients with two or fewer comorbidities (RR=0.57; p<0.001) and patients under the 75-year-old group (RR=0.53; p< 0.05). Conclusions: Hematologic malignancy, age, and the number of comorbidities are predictor factors for worse prognosis in COVID-19 infection.", "keywords": [ "COVID-19", "Cancer", "outcome", "oncology", "Indonesia" ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 It is shown that 2.1% of patients with confirmed COVID-19 were reported also to have cancer.2 A meta-analysis revealed that the mortality rate for COVID-19 patients with cancer was 21.1%. As many as 45.4% of cancer patients with COVID-19 have severe or critical symptoms.2\n\nCancer patients are a uniquely susceptible population because most of these patients may be in a suboptimal physical condition while also requiring cytotoxic drugs that can reduce immunity. In addition to the symptoms of COVID-19, these patients' cancer treatment may also be delayed during the pandemic.3\n\nMost observational studies have exposed that COVID-19 patients with cancer tend to have worse prognosis compared to non-cancerous COVID-19 patients.4,5 Previous research revealed that patients with hematologic cancers (HC) experienced more severe COVID-19 symptoms and higher CFR (case fatality rate) side to those with solid cancers (SC).6 More severe manifestation and higher CFR are found in hematologic cancer patients compared to solid cancer patients.\n\nPrevious research has suggested that the presence of haematological malignancies may reduce COVID-19 severity progression due to an attenuated inflammatory response.7–9 Other studies have reported that solid tumors were a worse prognosis predictor.10,11 The variation between studies and the lack of publications have encouraged us to analyze if patients with hematologic cancer and those with solid tumors would fare differently in the setting of COVID-19 infection.\n\n\nMethods\n\nThe Preferred Reporting Items for Systematic Review and Meta-Analysis Protocol PRISMA) guidelines were used to guide this study.22\n\nThis review included clinical studies (clinical trial, retrospective, or prospective) of all cancer patients who had COVID-19 infection based on polymerase chain reaction (PCR) test. Articles published between December 2019 to January 2021 in English were considered. For inclusion, the published articles must have had documentation of COVID-19 infection in both solid cancer and hematological cancer patients. Proceeding, commentaries, and editorials without a peer-review process were excluded.\n\nWe systematically searched databases to identify eligible articles using PubMed and Google Scholar for articles published from December 2019 to January 2021 using the search strategy in Table 1. We also researched references lists of relevant articles to identify additional primary studies and minimize bias.\n\nAll articles from the search strategy were screened further for eligibility. The titles and abstracts were independently screened and reviewed by three authors (FP, AR, RM). The article's technical uncertainties were resolved through discussion between all authors (FP, AR, RM, JH, RI, IB, MJ). Study assessment was based on the following criteria: 1) published in English, 2) prospective or prospective study on cancer patients with COVD-19 infection; 3) sufficient data relating to PICO (participants/interventions/comparisons/outcomes) criteria (Table 2) from COVID-19 patients with hematological and primary solid cancer.\n\nCOVID-19=coronavirus disease 2019.\n\nThe data collected were demographic details (e.g., age, race, comorbidities), type of cancer (primary solid tumors and hematological malignancy), patient's anti-cancer therapies (described as surgery, chemotherapy, radiotherapy of immunotherapy), clinical outcomes (developing severe events, hospitalization rates, intensive care unit (ICU) admission rates, 30-days mortality rate and case-fatality rate). Three authors (FP, AR, RM) extracted the data, jointly reconciled, and discussed technical uncertainties. The authors then appraise the studies using the Newcastle-Ottawa scale (NOS) for cohort studies (Table 3).2\n\nThe statistical analysis, including Cochran–Mantel–Haenszel test (CMH), Risk Ratio, Heterogenicity test, and funnel plotting were done using Statistical Package for the Social Sciences (SPSS) 25, MedCalc Statistical Software version 19.3, and RevMan version 5.4.\n\n\nResults\n\nThe study selection for this review can be seen in Figure 1. In total, 20 articles were included in the analysis.\n\n\nSystematic review\n\nIn 2020, DeMelo et al. reported that age, advanced malignancy stage, and number of metastases were associated with clinical fragility and higher risk of death in COVID-19 patients.10\n\nA previous study explained that COVID-19 symptoms in cancer patients ranged from mild symptoms (55% of cases), which required outpatient management only (fever, cough, fatigue, myalgia, etc.) to moderate to severe symptoms (45% of cases). From these patients, 42% were admitted to the ICU.10 According to a study, a complication such as secondary infection and acute respiratory distress syndrome occurred in a majority of cancer patients (63%).4 Another study in 2020 found that patients with malignancy were prone to having COVID-19 with severe manifestation (54% vs. 35%; P=0.003). The study mentioned that severe COVID-19 symptoms upon admission are a significant risk of in-hospital death (hazard ratio=28.2).11 Two other studies reported similar findings which support that cancer is associated with worse outcome.12,13\n\nRegarding type of cancer, Dai and colleagues reported the hematologic cancer and lung cancer group had the highest and second highest severity and death rates compared to other cancer groups. The patients with hematological malignancy have reduced immunity and are more prone to infection, which can exacerbate COVID-19 infection.8 Previous studies showed that leukemia, lymphoma, and myeloma as hematological cancer groups could increase death rate, ICU admission, critical manifestation, and invasive mechanical ventilation requirement.8\n\nA previous study showed that patients with and without cancer had similar COVID-19 severity. In the said study, the hematological and solid tumors groups showed non-significant trends for immediate manifestation of severe events (hematological group cohort = 30% vs. solid group cohort = 61.4%).16 However, another study from Canada investigated 252 cancer patients with COVID-19 and showed that 28% of adult patients had a high mortality rate, whereas none of the patients in the pediatric cohort had a significant illness. In hospital-acquired patients with COVID-19, overall survival (OS) was shorter than those with community-acquired infection.17 Similarly, a study from the UK reported that patients with hematological cancer have a greater risk of severe COVID-19 clinical manifestation, which needs more intensive supportive interventions and poses a greater risk of death than non-cancer patients.5 Tremblay and colleagues explained that the hematologic malignancies group of patients might be vulnerable to COVID-19. The preliminary study also suggests that hematological cancer patients have higher mortality than the general population.18\n\nDifferent cancer treatments including surgical, radiotherapy and COVID-19-specific medication done within 60 days before COVID-19 infection did not affect the death risk.10 Two studies reported an increased death rate in patients who received immunotherapy, surgery and chemotherapy.8,19 Robilloti demonstrated that lung cancer patients treated with immune checkpoint inhibitors (ICI) correlated with worse COVID-19 infection outcomes.17 On the other hand, patients with lung cancer who had COVID-19 had better outcomes despite having immunotherapy.13\n\nRivera et al. analyzed the treatments of COVID-19 in patients with cancer. High-dose corticosteroids combined with other therapies were correlated to higher mortality than positive and negative controls. Hydroxychloroquine combined with other drugs also demonstrated similar results, in which when combined, the risk of all-cause mortality every 30-day was increased when compared with the positive control (OR=2.15). On the other hand, remdesivir showed potential benefit as lower 30-day all-cause mortality compared to positive group (OR=0.41).19\n\n\nMeta analysis\n\nTable 4 shows a summary of the included studies.\n\nIn total, 14 studies included detailed case fatality rates of hematological cancer and primary solid cancer groups. Overall, the case-fatality rate in the hematological cancer group was 1.22 fold higher than the primary solid cancer group (263/976 vs. 852/4373; RR 1.22; CI 95% [1.08-1.37]; P<0.001) (Figure 2).\n\nCI=confidence interval, df=degrees of freedom, M-H=Mantel Haenszel method.\n\nWe performed two sub-analyses on case-fatality rate, to determine the correlation with comorbidities and age. Two studies provided data on the patients' comorbidites (two or less comorbidities and more than two comorbidities group).10,24 Overall, the case-fatality rate in patients with two or fewer comorbidities group was 0.57-fold lower than patients with more than two comorbidities group (97/694 vs. 65/327; RR 0.57; CI 95% [0.42-0.76]; P<0.001) (Figure 3). We also calculated the pooled proportion of case-fatality rate in the cardiovascular disease group (42.5%) (Figure 4), hypertension (36.8%) (Figure 5), and diabetes mellitus (36,8%) (Figure 6), as those three were deemed the most prevalent comorbidities.\n\nCI=confidence interval, df=degrees of freedom, M-H=Mantel Haenszel method.\n\nIn total, six studies included detailed data of elderly patients (under 75 y.o. and 75 y.o. or older) in both cancer groups. Overall, the rate of death in patients under 75 y.o. group was 0.53 fold lower than patients under 75 y.o. group (250/1350 vs. 154/465; RR 0.53; CI 95% [0.36-0.80]; P=0.002) (Figure 7).\n\nOverall, five studies included detailed data of patients who developed critical events in the hematological and primary solid cancer groups separately. Overall, the rate of critical care events in the hematological cancer group was 1.65 fold higher than the primary solid cancer group (140/371 vs. 585/2312; RR 1.65; CI 95% [1.22-2.23]; P=0.001) (Figure 8).\n\nCI=confidence interval, df=degrees of freedom, M-H=Mantel Haenszel method.\n\n\nDiscussion\n\nTo our best knowledge, the severity of COVID-19 can be worsened by cancer. The risk of death may also increase due to cancer. Patients with hematologic malignancy have an immunocompromised state which may induce co-infection and thus aggravate COVID-19 clinical presentation.4,5,8–14,17 Our meta-analysis shows that the rate of mortality and critical care events were higher in the hematologic group than in the primary solid cancer group. At the same time, the case-fatality is higher in patients who had more than two comorbidities and patients aged 75 or older. Thus, our analysis showed a tendency toward publication bias for case-fatality rate (P=0.03) (Figure 9) likely to the presence of small sample size studies.\n\nOur analysis on critical care events seemed to differ from the rest of the study. The COVID-19 diagnosis test might cause this as both PCR and anti-SARS-CoV-2 IgG/IgM antibody tests12 were used in Li's study, whereas other studies included in the meta-analysis only used PCR for diagnostic testing. Moreover, This was a retrospective study with relatively few subjects yet with an enormous number of controls.12\n\nThe hematological cancer group had more severe COVID-19 manifestation.12 However, this finding requires further verification through multi-center studies. Based on a previous study, delaying surgery or chemotherapy for patients with cancer during the COVID-19 pandemic is not required, especially in areas with fewer COVID-19 patients.16\n\nFrom our review, several studies from China, Europe, and North America reported that cancer patients with COVID-19 infection who received chemotherapy, immunotherapy, and ICI treatment had a higher death risk.4,5,8,17,20 A meta-analysis in the US reported that active cytotoxic chemotherapy was associated with a high risk of adverse outcomes from COVID-19.21 At the same time, Stroppa et al. revealed a better prognosis of COVID-19-infected lung cancer patients treated with immunotherapy.14 Similarly, Fillmore et al. reported a lower risk of infection was correlated with ICI treatment.14 A meta-analysis by Yekedüz et al. revealed that cancer treatment was not associated with severity and mortality risk of COVID-19 within the last 30 days before diagnosis.22\n\nA COVID-19 and Cancer Consortium Cohort Study in US revealed that corticosteroids in high dose administration combined with any other therapies, and hydroxychloroquine combined with other drugs or given alone were associated with higher 30-day all-cause mortality risk in cancer patients with COVID-19 infection. While remdesivir has shown to be a potential treatment, the all-cause mortality rate in 30 days decreases.19\n\nLimitations of this review include that the review section may have been influenced by the authors' personal viewpoints, gaps in literature searching practices may have led to the omission of relevant research, and errors in the translation of data from the primary literature to summarization in the review. There were also missing data points from some studies. Given these limitations, we encourage conducting multi-center registries (web/online-based) to obtain all the data from every individual case of cancer patients with COVID-19 infection.\n\n\nConclusion\n\nHematological malignancy, older age (75 years) and the number of comorbidities are predictors for worse prognosis in COVID-19 infection. The therapy protocol for cancer patients with COVID-19 infection and COVID-19 therapy is still debatable. Future research needs to evaluate these treatments in prospective randomized controlled trials (RCTs), address disparities, and promote studies evaluating potential anti-COVID-19 therapies.\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 'Comparison of Clinical Outcome in Hematological Cancer Compared to Primary Solid Cancer Patients With COVID-19 Infection: a Systematic Review and Meta-Analysis, https://doi.org/10.6084/m9.figshare.17122541.29\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nZheng J: SARS-coV-2: An emerging coronavirus that causes a global threat. Int J Biol Sci. 2020; 16(10): 1678–1685. PubMed Abstract \| Publisher Full Text\n\nElGohary GM, Hashmi S, Styczynski J, et al.: The risk and prognosis of COVID-19 infection in cancer patients: A systematic review and meta-analysis. Hematol Oncol Stem Cell Ther. July 2020; PubMed Abstract \| Publisher Full Text\n\nGavillet M, Carr Klappert J, Spertini O, et al.: Acute leukemia in the time of COVID-19. Leuk Res. 2020; 92: 106353. PubMed Abstract \| Publisher Full Text\n\nYang K, Sheng Y, Huang C, et al.: Clinical characteristics, outcomes, and risk factors for mortality in patients with cancer and COVID-19 in Hubei, China: a multicentre, retrospective, cohort study. Lancet Oncol. 2020; 21(7): 904–913. PubMed Abstract \| Publisher Full Text\n\nLee LYW, Cazier JB, Starkey T, et al.: COVID-19 prevalence and mortality in patients with cancer and the effect of primary tumour subtype and patient demographics: a prospective cohort study. Lancet Oncol. 2020; 21(10): 1309–1316. PubMed Abstract \| Publisher Full Text\n\nBaşcı S, Ata N, Altuntaş F, et al.: Patients with hematologic cancers are more vulnerable to COVID-19 compared to patients with solid cancers. Intern Emerg Med. 2021: 0123456789. PubMed Abstract \| Publisher Full Text\n\nVijenthira A, Gong IY, Fox TA, et al.: Outcomes of patients with hematologic malignancies and COVID-19: a systematic review and meta-analysis of 3377 patients. Blood. 2020; 136(25): 2881–2892. PubMed Abstract \| Publisher Full Text\n\nDai M, Liu D, Liu M, et al.: Patients with Cancer Appear More Vulnerable to SARS-CoV-2: A Multicenter Study during the COVID-19 Outbreak. Cancer Discov. 2020; 10(6): 783–791. PubMed Abstract \| Publisher Full Text\n\nRyuta S, Teiji T; for Malignant Gliomas: 2017; 8–16. Publisher Full Text\n\nDe Melo AC, Thuler LCS, Da Silva JL, et al.: Cancer inpatients with COVID-19: A report from the Brazilian National Cancer Institute. PLoS One. 2020; 15(10): e0241261–e0241215. PubMed Abstract \| Publisher Full Text\n\nFerrari BL, Ferreira CG, Menezes M, et al.: Determinants of COVID-19 Mortality in Patients With Cancer From a Community Oncology Practice in Brazil. JCO Glob Oncol. 2021; 7: 46–55. PubMed Abstract \| Publisher Full Text\n\nLi Q, Chen L, Li Q, et al.: Cancer increases risk of in-hospital death from COVID-19 in persons <65 years and those not in complete remission. Leukemia. 2020; 34(9): 2384–2391. PubMed Abstract \| Publisher Full Text\n\nMeng Y, Meng Y, Lu W, et al.: Cancer history is an independent risk factor for mortality in hospitalized COVID-19 patients: A propensity score-matched analysis. J Hematol Oncol. 2020; 13(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nStroppa EM, Toscani I, Citterio C, et al.: Coronavirus disease-2019 in cancer patients. A report of the first 25 cancer patients in a western country (Italy). Future Oncol. 2020; 16(20): 1425–1432. PubMed Abstract \| Publisher Full Text\n\nShoumariyeh K, Biavasco F, Ihorst G, et al.: Covid-19 in patients with hematological and solid cancers at a Comprehensive Cancer Center in Germany. Cancer Med. 2020; 9(22): 8412–8422. PubMed Abstract \| Publisher Full Text\n\nElkrief A, Desilets A, Papneja N, et al.: High mortality among hospital-acquired COVID-19 infection in patients with cancer: A multicentre observational cohort study. Eur J Cancer. 2020; 139: 181–187. PubMed Abstract \| Publisher Full Text\n\nRobilotti EV, Babady NE, Mead PA, et al.: Determinants of severity in cancer patients with COVID-19 illness. Nat Med. 2020; 26(8): 1218–1223. PubMed Abstract \| Publisher Full Text\n\nTremblay D, Seah C, Schneider T, et al.: Convalescent Plasma for the Treatment of Severe COVID-19 Infection in Cancer Patients. Cancer Med. 2020; 9(22): 8571–8578. PubMed Abstract \| Publisher Full Text\n\nRivera DR, Peters S, Panagiotou OA, et al.: Utilization of COVID-19 Treatments and Clinical Outcomes among Patients with Cancer: A COVID-19 and Cancer Consortium (CCC19) Cohort Study. Cancer Discov. 2020; 10(10): 1514–1527. PubMed Abstract \| Publisher Full Text\n\nFillmore NR, La J, Szalat RE, et al.: Prevalence and Outcome of COVID-19 Infection in Cancer Patients: A National Veterans Affairs Study. JNCI J Natl Cancer Inst. 2020; 113(August): 691–698. PubMed Abstract \| Publisher Full Text\n\nPark R, Lee SA, Kim SY, et al.: Association of active oncologic treatment and risk of death in cancer patients with COVID-19: a systematic review and meta-analysis of patient data. Acta Oncol. 2021; 60(1): 13–19. PubMed Abstract \| Publisher Full Text\n\nYekedüz E, Utkan G, Ürün Y: A systematic review and meta-analysis: the effect of active cancer treatment on severity of COVID-19. Eur J Cancer. 2020; 141: 92–104. PubMed Abstract \| Publisher Full Text\n\nAntrim L, Capone S, Dong S, et al.: Impact of COVID-19 infection among cancer patients treated at the Los Angeles County Medical Center. Cancer Treat Res Commun. 2021; 26: 100273. PubMed Abstract \| Publisher Full Text\n\nKuderer NM, Choueiri TK, Shah DP, et al.: Clinical impact of COVID-19 on patients with cancer (CCC19): a cohort study. Lancet. 2020; 395(10241): 1907–1918. PubMed Abstract \| Publisher Full Text\n\nJazieh A-R, Alenazi TH, Alhejazi A, et al.: Outcome of Oncology Patients Infected With Coronavirus. JCO Glob Oncol. 2020; 6: 471–475. PubMed Abstract \| Publisher Full Text\n\nRüthrich MM, Giessen-Jung C, Borgmann S, et al.: COVID-19 in cancer patients: clinical characteristics and outcome-an analysis of the LEOSS registry. Ann Hematol. 2021; 100(2): 383–393. PubMed Abstract \| Publisher Full Text\n\nWang QQ, Berger NA, Xu R: Analyses of Risk, Racial Disparity, and Outcomes among US Patients with Cancer and COVID-19 Infection. JAMA Oncol. 2020; 7: 220–227. PubMed Abstract \| Publisher Full Text\n\nde Joode K , Dumoulin DW, Tol J, et al.: Dutch Oncology COVID-19 consortium: Outcome of COVID-19 in patients with cancer in a nationwide cohort study. Eur J Cancer. 2020; 141: 171–184. PubMed Abstract \| Publisher Full Text\n\nAl Farabi MJ: PRISMA NS Checklist.pdf. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "124022", "date": "21 Mar 2022", "name": "Zubing Mei", "expertise": [ "Reviewer Expertise Systematic review", "prediction model. colorectal diseases", "surgery", "guideline." ], "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 systematic review study aimed to analyze the outcome of COVID-19 patients with hematological cancer and primary solid cancer worldwide. By combining 20 articles in the analysis, the author found that hematologic malignancy, age, and the number of comorbidities were predictor factors for worse prognosis in COVID-19 infection. I have major comments for the following points:\n1. The authors did not search the other two major databases: Embase and Cochrane Library, which may lead to publication bias.\n2. The search strategy is not adequate. Even for Pubmed, the author should adhere to the combination of Mesh and free text words search.\n3. A detailed literature screening process was not presented.\n4. Statistical analysis is far from adequate. How did the author treat heterogenicity, by subgroup analyses or meta-regression? Have they tested for publication bias or sensitivity analysis? The result should be presented in the 'Results' section instead of the 'Discussion' section.\n5. In Figure 2, the pooled RR is not consistent with the report in the 'Results' section.\n6. The Discussion section is poorly reported. What are the strengths of this study? How did the findings impact clinical practice?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "124023", "date": "22 Mar 2022", "name": "Sri Maliawan", "expertise": [ "Reviewer Expertise neuroscience" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. This study entitled with clinical outcomes but in your paper, there aren't any explanation regarding the critical events.\n2. Regarding the outcome for this study, are there any time similarity to evaluate it?\n3. Please clarify the incidences and causes of deaths in each study.\n4. Figure 2 showed different RR with the text mentioned above it. In the text, the was RR 1.22 but the figure showed 1.23, also the lowest internal was 1.08 in the text, but the figured showed 1.09.\n5. Regarding the conclusion, does \"PROGNOSIS\" stand for \"CLINICAL OUTCOME\" in this study?\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? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-202
https://f1000research.com/articles/10-369/v1	10 May 21	{ "type": "Software Tool Article", "title": "Bayes Lines Tool (BLT): a SQL-script for analyzing diagnostic test results with an application to SARS-CoV-2-testing", "authors": [ "Wouter Aukema", "Bobby Rajesh Malhotra", "Simon Goddek", "Ulrike Kämmerer", "Peter Borger", "Kevin McKernan", "Rainer Johannes Klement", "Bobby Rajesh Malhotra", "Simon Goddek", "Ulrike Kämmerer", "Peter Borger", "Kevin McKernan" ], "abstract": "The performance of diagnostic tests crucially depends on the disease prevalence, test sensitivity, and test specificity. However, these quantities are often not well known when tests are performed outside defined routine lab procedures which make the rating of the test results somewhat problematic. A current example is the mass testing taking place within the context of the world-wide SARS-CoV-2 crisis. Here, for the first time in history, laboratory test results have a dramatic impact on political decisions. Therefore, transparent, comprehensible, and reliable data is mandatory. It is in the nature of wet lab tests that their quality and outcome are influenced by multiple factors reducing their performance by handling procedures, underlying test protocols, and analytical reagents. These limitations in sensitivity and specificity have to be taken into account when calculating the real test results. As a resolution method, we have developed a Bayesian calculator, the Bayes Lines Tool (BLT), for analyzing disease prevalence, test sensitivity, test specificity, and, therefore, true positive, false positive, true negative, and false negative numbers from official test outcome reports. The calculator performs a simple SQL (Structured Query Language) query and can easily be implemented on any system supporting SQL. We provide an example of influenza test results from California, USA, as well as two examples of SARS-CoV-2 test results from official government reports from The Netherlands and Germany-Bavaria, to illustrate the possible parameter space of prevalence, sensitivity, and specificity consistent with the observed data. Finally, we discuss this tool’s multiple applications, including its putative importance for informing policy decisions.", "keywords": [ "Bayes", "COVID19", "PCR Test", "SARS-CoV-2", "SQL" ], "content": "1. Introduction\n\nIn December 2019, a cluster of patients with pneumonia of unknown origin was associated with the emergence of a novel beta-coronavirus of bat origin,1 first named 2019-nCoV2 and later specified as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2).3 This outbreak led to the rapid development of reverse transcriptase - quantitative polymerase chain reaction (RT-qPCR) tests to identify SARS-CoV-2 RNA in specimens obtained from patients.2,4\n\nAfter sporadic SARS-CoV-2 positive cases in January5,6 to the end of February 2020 worldwide cases of the SARS-CoV-2-associated disease COVID-19 began to accumulate, causing policymakers in many countries to introduce countermeasures. These Non-Pharmaceutical Interventions (NPIs) predominantly started worldwide around March 2020 while the virus was characterized as a pandemic on 11 March, 2020.6,7 As a result, for more than one year now, large parts of the world are in a COVID-19 crisis-mode with daily reporting of SARS-CoV-2 cases in dashboards worldwide.8 The definition of cases and prevalence estimates was based on RT-qPCR testing, independent of the clinical diagnosis. Thereby, a person is considered a case (i.e., infected), once a test turns out positive.9\n\nLike all laboratory tests, however, the SARS-CoV-2 RT-qPCR tests are not flawless. This is because sensitivity and specificity depend on a multiplicity of confounding factors. These factors cover the test design, the lab application, and possible contaminations with substances/nucleic acids interfering with the reaction.10,11 Consequently, both false-negative and false-positive results have been reported.12,13 Nevertheless, the test system’s limitations are rarely discussed in scientific publications and public health systems despite their crucial role for making inferences about the possible infection status of a tested person.14 Many more or less defined commercial and laboratory ‘in house’ tests are now routinely being used,15 often without standardised guidelines, which leads to entirely unknown test performance specifications.16 The few studies aiming to estimate sensitivity and specificity of SARS-CoV-2 RT-qPCR tests have reported sensitivities and specificities in the ranges ≳30% and ≳80%, respectively - therefore, the communicated data seldom can offer precise distinctions.14\n\nGiven the critical role that dashboards and graphs based on SARS-CoV-2 test results play for policymakers, health professionals, and the general public,8 our objective was to develop a Bayesian calculator that could calculate test quantities and prevalence solely based on officially reported numbers of total and positive tests, i.e., without making any a priori assumptions. In this way, time trend estimates and country-to-country comparisons of these test performance measures as well as disease prevalence estimates become possible, producing in-depth insights, making projections/simulations possible, and providing a more holistic understanding of the daily incoming data in general.\n\n\n2. Methods\n\nThe Bayes Lines Tool (BLT) calculator is based on Bayes’ theorem and estimates the true and false positive, and true and false negative numbers at a given time point for which the total number of tests performed and the number of positive test results is known. These data are usually reported and published by official government bodies daily and/or weekly. Thus, the model uses the following information:\n\n• Publishing date or report identifier of the test data\n\n• Number of performed tests (#tests)\n\n• Number of reported positive results (#positives)\n\nThe model takes this information as a given fact and uses it to make inferences about the test performance parameters (sensitivity and specificity) as well as the prevalence (also known as the base rate) - these inferences are essential for estimating the number of true positives (TP), false positives (FP), true negatives (TN) and false negatives (FN). It is assumed that there is no knowledge of either the prevalence or the sensitivity and specificity of the tests used. Instead, the model explores all possible combinations of two of these three parameters within reasonable ranges specified by the user; for each of these combinations, the third parameter can then be calculated using the dependencies through Bayes’ theorem. Finally, all parameter combinations that result in TP+FP estimates consistent with the known number of positive tests are selected and stored as Bayesian confusion matrices (CMs).\n\nA single CM contains TP, FP, TN, and FN in absolute numbers ( Table 1). For a given prevalence, sensitivity, and specificity these are derived from Bayes’ theorem:\n\nHere, T denotes the hypothesis that a test comes out positive ( ¬T its denial) and I the hypothesis that an individual is infected, so that PI is the prevalence and PTI is the test sensitivity. PT is the marginal probability of a positive test, which we estimate as the frequency of positive test results, whereas PIT is the probability of being infected given that the test came out positive. With the normalizing constant PT estimated as PT=#positives#tests and PIT estimated as the proportion of infected individuals among those in which the test came out positive, equation (1) becomes:\n\nEquation (2) thus shows that the number of TPs depends on the prevalence, test sensitivity and total number of tests performed. Using P¬T¬I =specificity and #negatives = #tests−#positives, an analogous derivation leads to\n\nFrom Equations (2) and (3), FP and FN follow as\n\nFor the implementation presented here, the two parameters which varied are as follows:\n\n• Sensitivity from 0.005 to 1 with 0.005 increments.\n\n• Specificity from 0.005 to 1 with 0.005 increments.\n\nFor a given sensitivity and specificity as well as number of tests and positives, the prevalence can then be computed as\n\nHereby, calculations for combinations of sensitivity and specificity that add to ≤1 are omitted, and cases in which prevalence turns out negative or larger than 1 are discounted as unphysical.\n\nWe developed an SQL query that generates all possible Bayesian CMs for a series of diagnostic test results, without making assumptions about prevalence, sensitivity, or specificity.\n\nThe code in PostgreSQL is given as follows (Code 1):\n\n\n\nGiven the test results published in the databases and given all generated permutations and consequently all possible CMs, only those are returned that match the positive test results. With only the resulting CMs for which TP+FP match the positives reported in the input data, we are able to identify patterns that provide additional insights for further investigation.\n\nIn order to produce CMs for a series of reports, such as daily test result numbers, several approaches are possible. In this manuscript we describe a practical application for using a Batch/Script approach. The Script is used on Apple OSX, the example below using COVID-19 data from the Netherlands (Code 2):\n\n\n\nFor the examples demonstrated in the Results section below, we extracted test data from:\n\n- A hypothetical scenario used for assessing the performance of BLT and demonstrating the so-called spectrum effect17,18\n\n- Influenza data for the Californian Bay Area obtained from the California Open Data Portal at\n\nhttps://data.ca.gov/dataset/influenza-surveillance/resource/d2207905-14eb-4264-9a02-8b6ac15ddc39?inner_span=True\n\n- The Netherlands/Dutch Corona Dashboard database, used as examples for a daily report and a time trend analysis:\n\nhttps://coronadashboard.rijksoverheid.nl/landelijk/positief-geteste-mensen\n\n- The German LGL Bayern database, derived from RKI (Robert Koch Institute) data: https://www.lgl.bayern.de/gesundheit/infektionsschutz/infektionskrankheiten_a_z/coronavirus/karte_coronavirus/\n\n\n3. Results\n\nIn the following section examples are provided that demonstrate the application of our calculator for the data referenced in Section 2.3.\n\nConsider the following hypothetical scenarios displayed in Table 2 that we used for a general check of BLT’s performance. In scenarios 1 and 2, we consider a disease which has a prevalence of 20% in two different subpopulations (e.g. young and old people, respectively). Each subpopulation has its own test characteristics: In subpopulation 1, test sensitivity is 95% and specificity 75%, while in subpopulation 2, sensitivity is 75% and specificity 95%. Consider that 10,000 tests have been performed in the total population. In scenario 1, the total population consists of an equal mix of both subpopulations, while in scenario 2 the total population consists of 75% subpopulation 1. The different mixture of subpopulations leads to a different number of positive test results, and hence a different input for BLT. The overall test performance measures (sensitivity and specificity) are a weighted average between the subpopulation test performance measures. This is called the spectrum effect.17\n\nNow consider a different scenario, in which the total population is a mix between two subpopulations with different susceptibility towards the disease, and hence different prevalence, but the test performs equally well in both subpopulations. In scenario 3, each subpopulation contributes 50% to the overall population, while in scenario 4, the less susceptible population contributes 80% (8,000 tests). Now the overall prevalence is the weighted average of the subpopulation prevalence values, and overall test sensitivity and specificity are equal to those of the subpopulations.\n\nFigure 1 displays all solutions that BLT delivers for scenarios 1-4, with the known solutions of the overall and subpopulations highlighted. It is visible that the spectrum effect observed in Table 2 is also visible in Figure 1, as it translates into the percentages of TPs, TNs, FPs and FNs. What is critical is the fact that BLT, which only works with the total number of tests and positives obtained, would not be able to distinguish between scenarios 1, 3 and 4. All three are compatible with the output set of CMs. One should thus keep in mind for the interpretation of BLT’s output that the solution corresponding to reality is determined by the mix of subpopulations being tested, which in turn might have their own specific subpopulation prevalence, sensitivity and specificity values. In other words, one should be aware of the spectrum effect.17,18 If possible, one should thus use knowledge about prevalence and test performance measures to filter out the CMs consistent with what is known about “the reality”.\n\nThe correct solutions corresponding to the overall and subpopulations of these scenarios are highlighted as large colored points, while all other solutions compatible with the number of tests and positives are shown in grey. For scenario 3, no exact match of prevalence, sensitivity and specificity to the TP and FP numbers could be obtained with the step sizes used in Code 1, so that we display the closest matches. %TP, %TN, %FP, %FN: Percentages of TP, TN, FP and FN numbers relative to the total number of tests performed.\n\nFigure 2 shows the results of applying BLT to weekly influenza test data from the Californian Bay Area, USA. The upper panel displays the number of positive tests reported over time, where the estimated number of TPs is overlaid in small dots (CMs) whose color represents the estimated prevalence (see legend on the right of Figure 2). Filters have been applied on specificity (95.0% - 100.0%) and sensitivity (80.0% - 100.0%). One could see that the number of TP tests is close to number of positives reported, except for some deviations during the spring and summer months when prevalence was estimated correctly as low.\n\nThe view is filtered on specificity and sensitivity. The specificity filter ranges from 95.0% to 100.0%. The sensitivity filter ranges from 80.0% to 100.0%.\n\nThe lower panel shows the positive predictive value (PPV), for each CM, defined as PPV=TPTP+FP , which confirms a high accuracy of the tests: The median PPV of all CMs over time was almost 90%.\n\n269 daily reports were downloaded from the Dutch government Corona dashboard and processed with the SQL-query. This resulted in 809,830 CMs matching the daily reports from June 1st, 2020 until Feb 24th, 2021. The upper panel of Figure 3 plots the median PPV, with the corresponding number of performed and positive tests plotted in the lower panel. Note that the left and right y-axes in the lower panel are on different scales.\n\nNo filters on prevalence, specificity or sensitivity were applied here.\n\nIt can be observed that in contrast to the influenza example ( Figure 2), the PPVs are now much lower, with a median average around 50%. For this estimation, no filters were applied on sensitivity, specificity or prevalence. When a posteriori knowledge is available about the diagnostic tests and/or the circumstances in which they were performed, different scenarios can be applied to the output. This is exemplarily visualized in Figure 4, in which some reasonable filters for a SARS-CoV-2 testing environment have been applied. Notice how the PPV started to increase sharply from a median around 50% before mid-September 2020 to 80-90% during the fall and winter.\n\nThe example above shows the 40,200 possible CMs that fit the given report, for 90.0% ≤ sensitivity ≤ 99.9% and 95.0% ≤ specificity ≤ 99.5% and 0 ≤ prevalence ≤ 20%.\n\nFigure 5 shows the output of BLT applied to weekly SARS-CoV-2 testing data from Bavaria in Germany. The thick grey line displays the number of positive tests reported over time, while the colored batches show the solutions of BLT for the TP numbers according to prevalence. Note that in low prevalence scenarios, the TPs do usually not come close to the reported number of positives. At the end of the summer, the prevalence values compatible with the official test reports suggested low prevalence, but also a discrepancy between the number of positive tests and TPs, suggesting a large number of FPs.\n\nFor the true positives, the color shows details about prevalence. The Specificity filter ranges from 75.0% to 100%. The Sensitivity filter ranges from 30.0% to 100.0%. Reports range from week ending 26th February 2020 until week ending 17th February 2021.\n\n\n4. Discussion\n\nThe developed Bayesian calculator tool allows the estimation of possible values for the essential variables’ prevalence, sensitivity, and specificity for a specific period of time (e.g., daily or weekly, depending on the input data the user supplies). The solutions provided by BLT are derived from Bayes’s theorem ( Equation 1) under the assumption that PT=#positives#tests and PIT=TP#positives . In cases of low total and positive test numbers, these assumptions might not hold exactly, but BLT should nevertheless find close solutions to the actual test performance measures. As our applied examples show, the strength of BLT lies in its application to mass testing scenarios such as those conducted during the SARS-CoV-2 crisis.\n\nThe BLT calculations are unbiased in the sense that they use all possible and sensible combinations of prevalence, sensitivity, and specificity, and let Bayes’ theorem decide which combinations match the actually observed data. The result for a given matching combination of these three particular parameters is provided in the form of a CM which contains the TP, TN, FP, and FN numbers. In the case where more than one combination is compatible with the given input data, the user may start simulating different scenarios, e.g., by applying prior knowledge regarding the expected prevalence range on a given date. This enables the user to further constrain the combinatorial possibilities of the output variables.\n\nPrevalence is a crucial factor for any inferences based on diagnostic tests, even though it is often not taken into account in practice. This results in the so-called base-rate fallacy.19 Our calculator may result in several possible prevalence values that are compatible with the observed data. In this case, knowledge about the population that has been tested may be used to constrain the possibilities. In 2020, for instance, prevalence-values in the range 12-15% were estimated for German hotspot regions,20,21 while prevalence was zero in an asymptomatic German mother-and-child population tested in April 2020.22 In an early COVID-19 related publication which compared RT-qPCR to chest computer tomography in 1014 COVID-19 patients from the Tongiji hospital in Wuhan, China, prevalence appeared to be very high: in total 830 patients were described to be confirmed or highly likely to have COVID-19, and of those 580 were diagnosed by chest CT and RT-qPCR and another 250 by CT and clinical decision. These results suggest a prevalence of 81.9% in these patients. A recent preprint publication23 aimed at estimating the sensitivity and specificity of the Chinese RT-qPCR tests by a Bayesian model incorporating information from both chest CT and clinical decision classification. The author obtained sensitivity of 0.707 (95% CI range: 0.668-0.749) and specificity of 0.851 (95% CI range: 0.774-0.941). Applying BLT to these data and assuming that only the cases in which both chest CT and RT-qPCR came out positive (i.e., filtering on 580 TPs), our model reveals a sensitivity of 65.3% and specificity ranging from 83.1%-83.6%, not too different from the estimates of the more complex analysis.23\n\nDuring the SARS-CoV-2 crisis an unprecedented mass testing not only of symptomatic, but also asymptomatic cases emerged as a strategy. One would expect the prevalence to be substantially higher in the former than in the latter population. As our scenarios 3 and 4 from section 3.1 shows, if there is a mixture of two populations with very different prevalence values, the resulting overall prevalence is a weighted average, provided that the sensitivity and specificity of the tests is similar in both populations.\n\nOur results confirm the recent World Health Organization (WHO) statement “that disease prevalence alters the predictive value of test results; as disease prevalence decreases, the risk of false positive increases”.24 This means that the probability that a person who has a positive result (SARS-CoV-2 detected) is truly infected with SARS-CoV-2 decreases as prevalence decreases, irrespective of the claimed specificity of the test system.24 This statement may be more accurately described as the number of TPs decreasing relative to a constant FP rate so the ‘risk of false positives’ only increases relative to the TP numbers, but the FP frequency is assumed to remain constant across a given number of tests. However, multiple modes of error may be in play. We should not assume FPs are independent of contamination from TP samples. There are higher risks of contamination in rapidly growing laboratories. Contamination of samples in the low disease prevalence seasons (summer) will go unnoticed as they do not produce a qPCR signal. Contamination prone methods may only become evident in the form of elevated and perhaps falsely assumed TPs once the disease prevalence increases in the winter.\n\nIn light of the current WHO statement, the rationale for mass testing strategies implemented during periods of low prevalence (e.g., summer) appears questionable. Furthermore, mass testing increases the risk of poor sample handling and laboratory contamination which might partly explain the high FP numbers our calculator predicts. For example, Patrick et al. argued that besides intrinsic test performance, amplicon contamination due to high throughput processing of samples within a laboratory would be the best explanation for an increased rate of FP detections made during an outbreak of the human coronavirus HCoV-OC43 in a Canadian facility.25\n\nWhile much attention has been placed on population frequency of disease and its impact on false positives, it is critical to understand the role of false negatives and the impact these can have on track and trace systems. The nasal swabs are known to vary tremendously in RNaseP Ct values suggesting highly variable sampling or limited RNA stability in the testing reagent chain.26 Woloshin et al. demonstrate 27-40% FNs with nasopharyngeal and throat swabs respectively and underscore the importance of understanding pre-test probabilities when interpreting qPCR results.27 These FN numbers are probably not due to the PCR itself, for which sensitivity is almost 100% (https://www.finddx.org/covid-19-old/sarscov2-eval-molecular/), but a matter of handling issues and the above discussed problems.\n\nWith the script presented here, we can think of many variations when it comes to the size/number of permutations, its step-size (granularity) and the ‘where’ clause as well as the strictness of matching TP+FP against the reported positives. For example, one could also increment prevalence on a log-scale to account for the fact that prevalence in many settings of diseases is very low.14\n\nWe are aware that choices made in these areas have a significant impact on the number of matching CMs. An impact/sensitivity analysis was not performed, although we suspect that such analysis might reveal additional insights. However, we think that the amount of matching CMs per result that the above query produces delivers sufficient material to make useful observations.\n\nFuture research with different data-repositories, for instance ECDC/TESSy-data would be very beneficial to identify a solid balance between precision (step-size in the permutations), number of matching CMs, and overall query performance.\n\n\n5. Conclusions\n\nWe have developed an easy-to-use Bayesian calculator (Bayes Lines Tool, BLT) to estimate prevalence, sensitivity, and specificity, and therefore TP, TN, FP, and FN numbers, from official test outcome numbers. With typical reports - especially as produced for SARS-CoV-2 tests - revealing just the number of positives and number of tests performed, the BLT SQL implementation generates CMs that fit within the boundaries of a typical simplified report, based on permutations of sensitivity and specificity. Its implementation is thereby not limited to SQL but can be applied on any platform of choice.\n\nThe ability to assess posterior probability independent of the circumstances in which diagnostic tests are performed, reveals a wide spectrum of opportunities for new applications both for the scientific community as well as for health professionals and policy makers around the globe. This is especially relevant for the mass testing taking place within the containment strategies of worldwide governments against the SARS-CoV-2. The BLT SQL query for the first time allows one to display a real estimation of the SARS-CoV-2 situation against the background of testing volume and quality and thus will provide a valuable tool for decision makers to monitor the test strategy and the effect of interventional procedures.\n\nThis tool will not only allow official institutions to survey the test situation and obtain a better basis for planning their interventions, but also allows for individuals who got tested to use the confusion matrices as an aid for interpreting their test results in view of the population they were tested in.\n\n\nData availability\n\nAll data underlying the results is linked in section 2.3 of the article. The hypothetical example is given in Table 2. No additional source data is required.\n\n\nSoftware availability\n\nZenodo:\n\nBayes Lines Tool (BLT) - A SQL-script for analyzing diagnostic test results with an application to SARS-CoV-2-testing, http://doi.org/10.5281/zenodo.4594210.28\n\nCode is available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe SQL-code and an example implementation in Excel and a Tableau work-book file can be downloaded at https://bayeslines.org/.", "appendix": "Acknowledgements\n\nWe thank Michiel Maandag for bringing down-to-earth counterweight and alignment to the team.\n\nWe wish to thank Dimitri Georganas for his support during the initial development of the model.\n\nFinally, we thank Andreas Macher for sharing his expertise in the optimisation of the SQL query.\n\n\nReferences\n\nRen LL, Wang YM, Wu ZQ, et al.: Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study. Chin Med J (Engl). 2020; 133(9): 1015–24.\n\nZhu N, Zhang D, Wang W, et al.: A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGorbalenya AE, Baker SC, Baric RS, et al.: The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020; 5(4): 536–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCorman VM, Landt O, Kaiser M, et al.: Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25(3): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWölfel R, Corman VM, Guggemos W, et al.: Virological assessment of hospitalized patients with COVID-2019. Nature. 2020; 581(7809): 465–9. PubMed Abstract \| Publisher Full Text\n\nHua J, Shaw R: Corona Virus (COVID-19) “Infodemic” and Emerging Issues through a Data Lens: The Case of China. Int J Environ Res Public Health. 2020; 17(7): 2309. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: WHO Director-General’s opening remarks at the media briefing on COVID-19-11 March 2020.2020 [cited 2021 Feb 6]. Reference Source\n\nEverts J: The dashboard pandemic. Dialogues Hum Geogr. 2020; 10(2): 260–4. Publisher Full Text\n\nEuropean Centre for Disease Prevention and Control: Case definition for coronavirus disease 2019 (COVID-19), as of 3 December 2020.2020 [cited 2021 Jan 22]. Reference Source\n\nvan Zyl G, Maritz J, Newman H, et al.: Lessons in diagnostic virology: expected and unexpected sources of error. Rev Med Virol. 2019; 29(4): 1–7. PubMed Abstract \| Publisher Full Text\n\nYounes N, Al-SAdeq DW, Al-Jighefee H, et al.: Challenges in Laboratory Diagnosis of the Novel. Viruses. 2020; 12(6): 582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWernike K, Keller M, Conraths FJ, et al.: Pitfalls in SARS-CoV-2 PCR diagnostics. Transbound Emerg Dis. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArevalo-Rodriguez I, Buitrago-Garcia D, Simancas-Racines D, et al.: False-negative results of initial RT-PCR assays for COVID-19: A systematic review. PLoS One. 2020; 15(12): e0242958. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlement RJ, Bandyopadhayay PS: The Epistemology of a Positive SARS-CoV-2 Test. Acta Biotheor. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMascuch SJ, Fakhretaha-Aval S, Bowman JC, et al.: A blueprint for academic laboratories to produce SARS-cov-2 quantitative RT-PCR test kits. J Biol Chem. 2020; 295(46): 15438–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou H, Liu D, Ma L, et al.: A SARS-CoV-2 Reference Standard Quantified by Multiple Digital PCR Platforms for Quality Assessment of Molecular Tests. Anal Chem. 2020; 93(2): 715–21. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSantos-Hövener C, Neuhauser HK, Rosario AS, et al.: Serology- And PCR-based cumulative incidence of SARS-cov-2 infection in adults in a successfully contained early hotspot (CoMoLo study), Germany, May to June 2020. Euro Surveill. 2020; 25(47): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReisinger EC, Von Possel R, Warnke P, et al.: Screening of Mothers in a COVID-19 Low-Prevalence Region: Determination of SARS-CoV-2 Antibodies in 401 Mothers from Rostock by ELISA and Confirmation by Immunofluorescence. Dtsch Medizinische Wochenschrift. 2020; 145(17): E96–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPadhye NS: Reconstructed diagnostic sensitivity and specificity of the RT-PCR test for COVID-19. medRxiv. 2020. Publisher Full Text\n\nWorld Health Organization: WHO Information Notice for IVD Users 2020/05.2021 [cited 2021 Jan 22]. Reference Source\n\nPatrick DM, Petric M, Skowronski DM, et al.: An outbreak of human coronavirus OC43 infection and serological cross-reactivity with SARS coronavirus. Can J Infect Dis Med Microbiol. 2006; 17(6): 330–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDahdouh E, Lázaro-Perona F, Romero-Gómez MP, et al.: Ct values from SARS-CoV-2 diagnostic PCR assays should not be used as direct estimates of viral load. J Infect. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoloshin S, Patel N, Kesselheim AS: False Negative Tests for SARS-CoV-2 Infection — Challenges and Implications. N Engl J Med. 2020; 383(6): e38. PubMed Abstract \| Publisher Full Text\n\nAukema W, Kämmerer U, Borger P, et al.: Bayes Lines Tool (BLT) - A SQL-script for analysing diagnostic test results with an application to SARS-CoV-2-testing (Version 4.2). Zenodo. 2021, March 10. Publisher Full Text" }	[ { "id": "84995", "date": "18 May 2021", "name": "Phillip M. Bentley", "expertise": [ "Reviewer Expertise Physics", "data analysis", "simulations", "computer modelling", "game theory", "strategy." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 a great job and this article should be indexed after addressing the issues below:\nEquation 6 has a typing mistake.\n\nThe authors state on page that \"These FN numbers are probably not due to the PCR itself, for which sensitivity is almost 100% (https://www.finddx.org/covid-19-old/ sarscov2-eval-molecular/), but a matter of handling issues and the above-discussed problems.\" This is incorrect. The false negative rate of the PCR test is documented as a function of time (see ref 1),1 and is mostly related to low early virus shedding initially. The middle phase, when the test has the lowest false negative rate, is as the authors describe, but then the lack of virus particles as the patient recovers becomes the major factor. Laboratory-based validation of PCR testing, as cited by the authors, differs from the clinical FN as seen in the present study due to potential flaws in the entire sample collection, handling, and processing chain. It is important to consider the whole chain in evaluating error rates. This is perhaps the most important aspect of test errors, since FP results in some inconvenience and/or worry whilst FN provides a dangerous false sense of security.\n\nThe analysis of Bavaria (figure 5) is roughly consistent with the numbers reported by Kucirka, but those of the Netherlands (figure 4) suggest a sensitivity range that is highly optimistic. The specificity on the other hand could be very good depending on the regional expertise.\n\nThe authors need to address the difference between their data and what would be expected from Kucirka et al.\n\nThe negative predictive value is very important for governments/regions to release healthy people with confidence. The authors should present this number as they have done for the positive predictive value.\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": "6803", "date": "18 Jun 2021", "name": "Rainer Klement", "role": "Author Response", "response": "We thank Dr. Bentley for his time to evaluate our article and constructive comments that we think have led to further improvements. We hope that the revised version can now be approved. Below, we reply to each of Dr. Bentley’s points. The revisions to the text have been marked with track changes in MS Word. Equation 6 has a typing mistake. Answer: Thank you for pointing it out, we advised the F1000 team to correct it. The authors state on page that \"These FN numbers are probably not due to the PCR itself, for which sensitivity is almost 100% (https://www.finddx.org/covid-19-old/ sarscov2-eval-molecular/), but a matter of handling issues and the above-discussed problems.\" This is incorrect. The false negative rate of the PCR test is documented as a function of time (see ref 1),1 and is mostly related to low early virus shedding initially. The middle phase, when the test has the lowest false negative rate, is as the authors describe, but then the lack of virus particles as the patient recovers becomes the major factor. Laboratory-based validation of PCR testing, as cited by the authors, differs from the clinical FN as seen in the present study due to potential flaws in the entire sample collection, handling, and processing chain. It is important to consider the whole chain in evaluating error rates. This is perhaps the most important aspect of test errors, since FP results in some inconvenience and/or worry whilst FN provides a dangerous false sense of security. Answer: Thank you for pointing out the time factor relative to the time of infection and the study by Kucirka et al. We have changed the text into the following: “These FN numbers are probably not due to the PCR itself, but are related to handling issues and the above discussed problems, as well as the time point within the course of infection that the sample is taken. In a meta-analysis of clinical data, Kucirka et al. found that the probability of a FN test was 100% at day 1 of an infection with SARS-CoV-2 (prior to symptom onset), and then decreased to 38% (95% credible interval 18-65%) at the day of symptom onset down to its minimum of 20% (12-30%) three days after symptom onset, after which it rose again to 66% (54-77%) three weeks after the infection. Hence, according to these numbers, even in infected individuals sensitivities below 30% are possible, a range that we excluded in our analysis consistent with Klement and Bandyopadhyay. This points to additional problems when testing asymptomatic individuals, because in case that they are truly infected, a high number of FNs is going to result.” We try to avoid judgements such as yours (“since FP results in some inconvenience and/or worry whilst FN provides a dangerous false sense of security“), which may invoke subjective arguments regarding “inconveniences\". We acknowledge that this is the general perception about FN versus FP and will elaborate further on this, with your last comment / point. We appreciate that you point this out and will add an additional figure to prevent any suggestion of bias in the article. The analysis of Bavaria (figure 5) is roughly consistent with the numbers reported by Kucirka, but those of the Netherlands (figure 4) suggest a sensitivity range that is highly optimistic. The specificity on the other hand could be very good depending on the regional expertise. Answer: When reducing sensitivity towards a less optimistic range of 60-80%, we see no visible impact on PPV in figure 4, probably because sensitivity has no significant influence on PPV. This is now described in words in Section 3.3. The authors need to address the difference between their data and what would be expected from Kucirka et al. Answer: A brief discussion has been added, in line with the answer to your point above: “Hence, according to these numbers, even in infected individuals sensitivities below 30% are possible, a range that we excluded in our analysis consistent with Klement and Bandyopadhyay. This points to additional problems when testing asymptomatic individuals, because even if they are infected, the resulting FN numbers may provide a false sense of security.” The negative predictive value is very important for governments/regions to release healthy people with confidence. The authors should present this number as they have done for the positive predictive value. Answer: Thank you for pointing this out. We understand and agree that NPV should be presented as prominently as PPV and will do so by adding a new figure for the Netherlands, taking in consideration your remarks about the sensitivity range being set too optimistically. Notice how NPV remains relatively high throughout the entire time range. Median NPV over time does not drop below 90%, even after we reduce the range for sensitivity to as low as 60-80%." } ] } ]	1	https://f1000research.com/articles/10-369
https://f1000research.com/articles/11-197/v1	16 Feb 22	{ "type": "Case Report", "title": "Case Report: Fortuitous discovery of cor triatriatum sinister in a young adult", "authors": [ "Achour Asma", "Mohamed Kacem Ben Slima", "Chamtouri Ikram", "Aida Ammar", "Mabrouk Abdelali", "Jamel Saad", "Badii Hmida", "Ahmed Zrig", "Mejdi Ben Massoud", "Walid Mnari", "Mezri Maatouk", "Mohamed Kacem Ben Slima", "Chamtouri Ikram", "Aida Ammar", "Mabrouk Abdelali", "Jamel Saad", "Badii Hmida", "Ahmed Zrig", "Mejdi Ben Massoud", "Walid Mnari", "Mezri Maatouk" ], "abstract": "Cor triatriatum is an extremely rare cardiac congenital malformation characterized by subdivision of the left atrium into two chambers. This division is due to the presence of an abnormal fibromuscular membrane interrupted by one or more openings that communicate the two chambers. The size and number of those orifices is variable, affecting the age of onset of symptoms and their severity. Our case describes the fortuitous discovery of this pathology in a young adult in her forties, following the finding of atrial fibrillation during a preoperative assessment of a strangulated hernia. The cardiac ultrasound allowed in our case to invoke the diagnosis, confirmed thereafter with an angioscanner. Surgical resection was not required in our patient who was put on medical treatment involving a beta-blocker to reduce atrial fibrillation and an anticoagulant treatment to decrease the thromboembolic risk, with a good clinical evolution. It seems therefore interesting to consider this uncommon pathology which may be latent for a long time, manifesting after some physiological or pathological situations.", "keywords": [ "Cor triatriatum", "defect", "heart", "cardiac CT-scan", "sinister", "dexter", "case report" ], "content": "Case presentation\n\nA 44-year-old G5P5 white female with no medical or surgical history and with no cardiovascular risk factors, who was a teacher and a mother of five, consulted the emergency room for occlusive syndrome and acute abdominal pain. She had no particular pathological history particularly with no cardiovascular disease risk factors. The diagnosis of a strangulated umbilical hernia was made, requiring emergency surgery. Physical examination revealed an irregular tachycardia at 150 beats per minute, a blood pressure of 120/70 mmHg, with a normal respiratory and neurological examination. An electrocardiogram as part of the preoperative assessment showed the presence of persistent atrial fibrillation previously unknown.\n\nA cardiac echography was made showing a dilated left atrium containing a septum dividing it into two chambers. Continuous wave Doppler allowed to locate the orifice within the accessory membrane without significant pressure gradient. The left ventricle was not dilated with a preserved ejection fraction. A cardiac CT-scan confirmed the diagnosis and allowed the visualization of an accessory chamber measuring 11.5 cm in diameter (Figure 1), receiving all pulmonary veins, and communicating with the left atrium via a large single opening in the membrane, three centimeters in size (Figure 2). No thrombus was detected, and no signs of pulmonary hypertension (Figure 3).\n\nOpen surgical resection was not performed in view of the absence of obstructive symptoms and the absence of pulmonary congestion symptoms.\n\nAtrial fibrillation was treated with beta-blocker by injection of 5 to 10 mg (1 mg/min) of Atenolol (Tenormine®) followed by an oral dose of 50 mg. The patient was subsequently put on a vitamin K antagonist with an initial dose of warfarin of 4 mg orally once a day followed by a maintenance dose of 7 mg orally once a day for life. The patient was discharged after two weeks with a regular sinus rhythm and a good general state. At three months follow-up, the patient kept a regular sinus rhythm with no worsening of the intra-atrial pressure gradient on ultrasound control.\n\nOur case seems unique because of the latency of the symptomatology despite the occurrence of several factors of decompensation, in particular the five pregnancies, explained by the width of the communication within the intra-atrial septum estimated at 3 cm.\n\nIn fact, this pathology generally becomes symptomatic in adulthood following several physiological or pathological factors of decompensation1 or following the appearance of fibrosis or calcification of the accessory membrane orifice.2 It is therefore important to supervise asymptomatic non-operated patients, as in our case, by regular clinical and ultrasound examination to prevent possible decompensation.\n\n\nDiscussion\n\nCor triatriatum is a rare congenital heart defect, representing 0.1 to 0.4% of all cardiac malformations,1 which can be isolated, or in up to 80% of cases2,3 associated with many cardiac malformations, most commonly atrial septal defect, followed by a patent foramen ovale and left superior vena cava. Less frequently, it is associated with anomalous pulmonary venous return, mitral valve regurgitation, isolated pulmonary artery stenosis, and tetralogy of Fallot.2,3\n\nTwo forms have been described: Cor triatriatum Sinister (CTS) in which the fibromuscular septum sits in the left atrium, and Cor triatriatum Dexter (CTD), rarer than CTS, interesting the right atrium.2\n\nCor triatriatum sinister, which corresponds to the case reported, was first described by Church in 1868.4 The embryonic cause remains not clearly established, but the most widely approved is malin corporation theory which consists of failure of the common pulmonary vein to fuse with the left atrium creating a complete, incomplete, or fenestrated septum.5\n\nClassification of CTS includes 3 types: A, B and, C. The case described here matches the type A.2\n\nPatients commonly develop symptoms early in life,1 during childhood or adolescence, or later in adulthood, or being completely asymptomatic throughout the whole life, depending on the number and size of orifices in the membrane.1 The most severe presentations can cause neonatal respiratory distress with an increased mortality risk.1 However, adults with the disease are usually asymptomatic.2\n\nSome physiological and pathological situations including all causes of increased cardiac flow and venous return may precipitate symptoms. Pregnancy is a common example associated with tachycardia and increased cardiac flow may cause cardiac decompensation in asymptomatic patients.6 In this case, the patient was in her forties at diagnosis and already had five pregnancies without any incidents reported, which is explained by the large communication between the two chambers minimizing hemodynamic implications.\n\nA chest x-ray as initial investigation may find pulmonary congestion (Kerley B-lines, acute pulmonary edema, pleural effusion), cardiomegaly with left atrial enlargement and prominent pulmonary vessels.7\n\nECG is most likely normal or may show unspecific features such as right axis deviation, S1Q3 pattern, or atrial arrhythmia.1,2\n\nA cardiac ultrasound done for rhythm disorders is usually the circumstance of discovery. It allows visualizing the membrane, number, and size of the openings, to estimate the transmembrane pressure gradient, flow velocity and to detect complications (such as thrombosis) and associated malformations.2\n\nRecently, the diagnosis performance of this technique has been improved by the contribution of 3D reconstruction of transesophageal US scan which shows exact membrane morphology.8\n\nClassically, diagnosis is made by cardiac angiography. Nowadays diagnosis is confirmed by a CT or MR scan.2 Magnetic Resonance Imaging (MRI) has the advantage of a finer study by showing more details. CT scan offers better spatial resolution and the possibility of multiplanar reconstructions.\n\nTreatment depends on the clinical presentation. The conservative approach is preferred in asymptomatic presentations.9\n\nThus, asymptomatic patients without pressure gradient do not require medical treatment, but in case of signs of pulmonary overload a medical treatment is proposed in addition to the surgical treatment.1 Some successful catheter ablation assays were also reported.2\n\nMedical treatment targets hemodynamic stabilization, cardiac rhythm control and anticoagulation for patients with atrial fibrillation.7\n\nSurgery is the definitive treatment. It consists of atriotomy, excision of interatrial membrane, and correction of associated anomalies.1 The result of surgical repair seems to be excellent in most the cases.2\n\nOur case report demonstrates the latency of this pathology depending on the size of the accessory membrane orifice. Indeed, this pathology may remain asymptomatic for a long time with a risk of being revealed at an adult age following certain situations, by a cardiac decompensation or a thromboembolic complication. The width of the orifice within the accessory membrane explains in our case the delay of the diagnosis in spite of five pregnancies which might have been responsible for a cardiac decompensation.\n\nThe rarity of this pathology and the lack of specificity of the clinical signs could orient us wrongly towards more frequent pathologies such as mitral stenosis, but the diagnosis is quickly rectified by the cardiac echography or secondarily by an angioscanner or an MRI. The prognosis after surgical treatment seem to be excellent according to a review of the literature,3 hence the interest of not delaying the positive diagnosis, avoiding the risk of sudden death to the patients.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nJha AK, Makhija N: Cor Triatriatum: A Review. Semin. Cardiothorac. Vasc. Anesth. 19 avr 2017; 21: 178–185. Publisher Full Text\n\nNassar PN, Hamdan RH: Cor Triatriatum Sinistrum: Classification and Imaging Modalities. Eur. J. Cardiovasc. Med. janv 2011 [cité 23 févr 2020]; 1(111). Publisher Full Text\n\nLambert J, Oechslin E, Tsang W, et al.: Cor Triatriatum Sinister with Secundum Atrial Septal Defect. CASE. août. 2017; 1(4): 141–146. PubMed Abstract \| Publisher Full Text\n\nChurch WS: Congenital malformation of the heart: abnormal septum in left auricle. Trans. PatholSocLond. 1868; 19: 188.\n\nLee ML: Concurrent cortriatriatum sinister and levoatriocardinal vein in an 11-year-old boy presenting with foudroyant pulmonary edema after appendectomy: a living tribute to the mal-incorporation theory. Anatol. J. Cardiol. 2018; 21: 172–174. PubMed Abstract \| Publisher Full Text\n\nBojanic K, Bursac D, Zmijanac D, et al.: Isolated cortriatriatumsinistrum and pregnancy: case report and reviewt of the literature.January 2013.\n\nAther B, Siddiqui WJ: Cor Triatriatum. StatPearls. Treasure Island (FL): StatPearls Publishing; 2020.\n\nPellicori P, Torromeo C, Boring Y: Cor triatriatum: Transoesophageal three-dimensional reconstruction shows exact membrane morphology. Arch. Cardiovasc. Dis. Mars. 2010; 103(3): 196–197. Publisher Full Text\n\ndos Santos FM , Flores LG, Dias FR, et al.: Cor Triatriatum Sinistrum in Symptomatic Adult: Case Report. Arq. Bras. Cardiol: Imagemcardiovasc. 2019; 32(1): 43–46." }	[ { "id": "127183", "date": "05 May 2022", "name": "Nagarajan Muthialu", "expertise": [ "Reviewer Expertise Paediatric cardiothoracic 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\nMany thanks for asking me to review this case report.\n\nWhile it is interesting to note the incidental finding and diagnosis of cor triatriatum in an adult, it is not a rare congenital defect warranting publication as a single case report.\n\nAdditionally (and more importantly), I have concerns on the diagnosis itself. The images do not show enough evidence of this being haemodynamically significant - the membrane is not shown fully on the slices of echocardiographic pictures. Even if they were to be shown, I wonder if conservative management is appropriate. The patient in question is in mid 40s age group. The risk of atrial arrhythmias, and consequent thromboembolic phenomenon will only worsen with time, and in the presence of this membrane, it is further accentuated, while we may not know the actual risk for this.\nWith these, I would think this is not appropriate for indexing in its current form.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "201245", "date": "11 Sep 2023", "name": "Ryohsuke Narui", "expertise": [ "Reviewer Expertise Atrial fibrillation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report described a patient with incidental discovery of cor triatriatum with atrial fibrillation. Treatment for cor triatriatum was not performed, and instead, the management focused on controlling the atrial fibrillation through rate control and anticoagulation therapy. While the impact of cor triatriatum was not significant, it is interesting that the left atrium was markedly enlarged.\nWhat is the mechanism of left atrial enlargement in this case? Please add this to the Discussion section.\n\nSince the CT scans shown in Figure 1-2 provide unclear left atrial structural details, please add a Figure of the CT scan with a 3D reconstruction of the left atrium.\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/11-197
https://f1000research.com/articles/11-194/v1	16 Feb 22	{ "type": "Case Report", "title": "Case Report: Cutaneous metastases indicative of endometrial cancer: A case report and review of the literature", "authors": [ "Olfa Zoukar", "Anis Haddad", "Yosra Jemaa", "Ines Zouari", "Rahma Issa", "Amel Bayar", "Ghada Khouildi", "Amal Sebri", "Dalel Naguez", "Asma Boukadida", "Mossaab Ghannouchi", "Raja Faleh", "Olfa Zoukar", "Anis Haddad", "Ines Zouari", "Rahma Issa", "Amel Bayar", "Ghada Khouildi", "Amal Sebri", "Dalel Naguez", "Asma Boukadida", "Mossaab Ghannouchi", "Raja Faleh" ], "abstract": "Secondary localizations of cutaneous metastases from endometrial cancer are rarely observed with a prevalence of 0.8% and can be indicative of deep pelvic cancer (ovarian or endometrial). The prognosis is usually poor, with skin metastases most often indicating advanced disease. This work, based on a case observed in our department and a review of the literature, aims to highlight the existence of this dramatic form of cutaneous extension of a common disease. Dermatologists are often consulted due to the non-specific nature of the lesions and should be aware of this entity. As with other cutaneous metastases, an accurate diagnosis is based on a the patient’s thorough medical and surgical history in conjunction with histopathology", "keywords": [ "cutaneous metastases", "endometrial cancer", "poor prognosis", "case report" ], "content": "Introduction\n\nEndometrial cancer is the third pelvic gynecological malignancy in Tunisia after breast and cervical cancer. It occurs most commonly in postmenopausal with 60 years of age being the frequency peak. However, up to 25% of cases can appear before menopause.1\n\nIts evolution is long and locoregional. Exceptionally, distant metastases to the abdominal wall, the lungs and bones can be revealing.\n\nCutaneous metastases are rarely observed with a prevalence of 0.8%.2 They most often present with an umbilical lesion, Sister Marie Joseph nodule. The spread of metastasis most often occurs via the lymphatic route, therefore close to the organ of origin, but it can also occur through the hematogenous route, therefore affecting distant organs or contiguity cutaneous metastases are typically painless when they are neither large nor infected. They have well-defined edges and are usually hard and attached to the subcutaneous tissues, covered with normal skin or slightly hyperemic (increased vascularization in the tumor) as long as the lesion does not become ulcerated. In the presence of cutaneous metastases, the prognosis is usually poor, with skin metastases most often indicating advanced disease. Almost half of these patients die within six months of the discovery of cutaneous metastases, the darkest prognosis accompanying cutaneous metastases from lung cancer.\n\nHereby, We report the case of a 61-year-old woman with an umbilical swelling which appeared six months prior to presentation secondarily ulcerated and oozing. She also reported an unintentional weight loss and a deterioration of her overall health with repetitive metrorrhagia. Biopsy of this lesion revealed the characteristics.\n\nA literature review is also provided.\n\n\nCase report\n\nA 61-year-old patient, house wife, with personal medical history of dyslipidemia and hypertension, obese with BMI 31, with no relevant family medical history, consulted her dermatologist for a budding peri-umbilical skin lesion that appeared six months before, initially neglected by the patient.\n\nThis clinical picture was associated with diffuse abdominopelvic pain with no colonic transit disorder. The patient reported unexplored repeated metrorrhagia. Physical examination showed a deterioration of the patient’s overall condition to 3 according to the WHO performance index, a painful, ulcerated and oozing umbilical nodule measuring 3 cm in diameter along its longest axis (Figure 1).\n\nIn addition, there was free ascites of low abundance and tumor-like hepatomegaly. The abdominal ultrasound revealed hetero-multi-nodular hepatomegaly of secondary appearance and ascites of low abundance. The umbilical skin lesion was biopsied and the pathology study concluded that there was a cutaneous metastasis of an adenocarcinoma whose histological appearance was compatible with a well differentiated endometrioid endometrial adenocarcinoma.\n\nThe patient was referred our department for additional treatment. On examination, the patient was apyretic, pale, asthenic, complaining of exertional dyspnea (walking perimeter of a few meters) and presented with a budding nodular skin lesion measuring 3 cm on the major axis of foul odor and bleeding in the biopsy area. The pelvic examinations revealed indurated rectovaginal septum with an enlarged uterus.\n\nBiological tests showed severe hypochromic microcytic anemia due to iron deficiency at 3.5 g/dl and tumor markers, in particular ACE, were increased. Pelvic ultrasound revealed a uterus of normal morphology but increased in size with a very thickened endometrium. The appendices were not seen with the presence of a low abundance effusion in the pouch of Douglas. The thoroco-abdominopelvic CT confirmed the ultrasound data with the presence of secondary lesions in the lungs and liver.\n\nA biopsy and hemostatic curettage of the endometrium was carried out after transfusion of 4 globular pellets and the anatomopathological study substantiated the presence of well differentiated endometrioid endometrial adenocarcinoma.\n\nAn abdominopelvic MRI performed as part of the extension assessment highlighted the presence of multiple pelvic iliac and lumboaortic lymphadenopathies with infiltration of nearby organs and secondary liver and digestive lesions.\n\nThe endometrial tumor was classified as T4B N2 M1 and the patient was non-operable and received 4 cycles of chemotherapy with pelvic external radiotherapy of 50 Gy. The evolution was unfavorable and death occurred 6 months later.\n\n\nDiscussion\n\nCutaneous metastases occur in 0.6% to 10.4% of all cancer patients. The frequency of Cutaneous metastases is correlated with the frequency of each malignant tumor, which is why women with skin metastases most often have the following primary malignant tumors: breast, ovary cavity, lung and large intestine. Globally, skin metastases represent 2% of all skin neoplasms.3–5\n\nThese lesions may be the only manifestation of an underlying visceral cancer.\n\nA systematic search of the English-language literature on PubMed between 1966 and 2013 identified only 26 cases of skin metastases in endometrial cancer.6,7 Most of these reports highlighted the rarity of this dissemination model.\n\nSkin metastasis of endometrial cancer is associated with a poor prognosis and an average life expectancy of approximately 4 to 12 months after diagnosis.8,9 It can take any form of lesion, including nodules, papules, ulcers and plaques. In our case, biopsy allowed us to confirm the presence of skin metastases related to endometrial cancer.\n\n\nConclusion\n\nOur patient represents a dramatic form of skin extension of a common disease. Dermatologists are often consulted due to the non-specific nature of the lesions and should be aware of this entity. As with other skin metastases, a thorough medical and surgical history in conjunction with histopathology is necessary for an accurate diagnosis.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nAuthor contributions\n\nAll the authors contributed to the conduct of this work. All authors also declare that they have read and approved the final version of the manuscript.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient before death and further consent was obtained from his husband after his death.", "appendix": "References\n\nDoghri R, Yahyaoui Y, Gabsi A, et al.: Les carcinomes de l’endomètre: étude anatomopathologique et histopronostique à propos de 62 cas dans un centre du Nord tunisien. Ann. Pathol. 38(2): 85–91. Publisher Full Text\n\nLerner LB, Andrews SJ, Gonzalez JL, et al.: métastases vulvaires secondaire à un carcinome à cellules transitionnelles de la vessie: un rapport de cas. J. Reprod. Med. 1999; 44: 729–732.\n\nLookingbill DP, Spangler N, Helm K: métastases cutanées chez les patients présentant un carcinomemétastatique: une étude rétrospective de 4020 patients. J. Am. Acad. Dermatol. 1993; 29: 228–236. PubMed Abstract \| Publisher Full Text\n\nAlcaraz I, Cerroni L, Rütten A, et al.: Les métastases cutanéesde tumeurs malignes internes: un examen clinico et immunohistochimique. Am. J. Dermatopathol. 2012; 34: 347–393. Publisher Full Text\n\nNashan D, Meiss F, Braun-Falco M, et al.: Les métastases cutanéesde malignités internes. Dermatol. Thérapie. 2010; 23: 567–580. Publisher Full Text\n\nGiordano G, Gnetti L, Melpignano M: Endométriale carcinome métastatique à la vulve: un rapport de cas et revue de la littérature. Pathol. Res. Pract. 2005; 201: 751–756. Publisher Full Text\n\nDamewood MD, Rosenshein NB, Grumbine FC, et al.: Cutanémétastases de cancer de l'endomètre. Cancer 1980; 46: 1471–1475. Publisher Full Text\n\nElit L, Lukka H, Friedman E: métastases cutanées du cancer de l'utérus papillaire séreux. Gynecol. Oncol. 2001; 82: 208–211. PubMed Abstract \| Publisher Full Text\n\nYilmaz Z, Bese T, Demirkiran F: métastases de la peau dans le cancer de l'ovaire. Int. J. Gynecol. Cancer 2001; 16(Suppl 1): 414–418. Publisher Full Text" }	[ { "id": "124048", "date": "07 Mar 2022", "name": "Fethi Derbel", "expertise": [ "Reviewer Expertise I am a professor of Surgery", "my area of research is mainly cancers" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 shows the delay in diagnosing an endometrial adenocarcinoma. The author describes the case report very well. The diagnosis delay was very well emphasised by the authors, however, the authors should give some recommendations for gynecologists and patients to make the diagnosis of endometrial adenocarcinoma earlier, before there is metastasis. The message in this article was mainly given to dermatologists to be aware of cutaneous metastasis.\nThe review of the literature concerning this article is up-to-date and the article is appropriate to be indexed without modifications.\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": "188624", "date": "17 Aug 2023", "name": "Ottavia D’Oria", "expertise": [ "Reviewer Expertise Gynecologic Oncology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with great interest the manuscript, which falls within the aim of this Journal and offers a high-quality overview of the topic. Although the manuscript can be considered already of high quality, I would suggest taking into account the following minor recommendations:\nI suggest another round of language revision, in order to correct a few typos and improve readability.\n\nThe introduction should be extended and completed. I find it interesting to include a reference to the molecular classification of endometrial cancer and to its implications to personalize the therapeutic path of patients with EC (see PMID: 36979434 and PMID: 36833105).\n\nI also suggest authors to better organize the discussion section following this ideal structure: main findings of the study, strength and limitations of the study, implications and comparison with literature, and future directions.\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": [] } ]	1	https://f1000research.com/articles/11-194
https://f1000research.com/articles/11-193/v1	16 Feb 22	{ "type": "Study Protocol", "title": "Treatment profiles and trajectories surrounding the diagnosis of major depressive disorder: a research protocol for a Danish register-based study.", "authors": [ "Pernille Herold Jeberg", "Anne Marije Christina Overgaard Nielsen", "Merete Osler", "Marie Kim Wirum-Andersen", "Ramune Jacobsen", "Anna Birna Almarsdóttir", "Kristoffer Jarlov Jensen", "Janne Petersen", "Anne Marije Christina Overgaard Nielsen", "Merete Osler", "Marie Kim Wirum-Andersen", "Ramune Jacobsen", "Anna Birna Almarsdóttir", "Kristoffer Jarlov Jensen", "Janne Petersen" ], "abstract": "Background: Major depressive disorder (MDD) is a prevalent illness that causes significant suffering and expenses at the personal and societal levels. The disorder is subject to heterogeneity reflected by diverse clinical phenotypes and assorted responses to treatment. Research on MDD treatments have focused on one treatment at a time, however many patients receive several different treatments. Considering the number of available treatment options, we hypothesize that it is possible to identify clinically meaningful groups of patients based on their psychiatric treatment. The objective of this study is therefore to identify psychiatric treatment profiles and trajectories of patients with major depressive disorder and, for the identified profiles and trajectories, to assess clinical and sociodemographic factors. Method: The study will be a population-based register study of patients with major depressive disorder in the Danish National Patient Register between 2011 and 2015. Using latent class analyses, we will identify homogenous groups of patients based on their psychiatric treatment patterns. These patterns constitute psychiatric treatment profiles which will be identified at six time-intervals, from 1.5 years before to 3 years after diagnosis of major depressive disorder. By cross-tabulating the identified treatment profiles, we will establish psychiatric treatment trajectories. Patients sharing profiles and trajectories will be characterized. Discussion: Identification of psychiatric treatment profiles and trajectories based on an unsupervised learning algorithm have the potential to reveal hidden patterns of psychiatric treatment. This will potentially pave the way for future studies of treatment combinations and a larger insight into the different courses of treatment. Furthermore, the assessment of clinical and sociodemographic factors may indicate different patient characteristics across treatment profiles and trajectories.", "keywords": [ "Denmark", "Depression", "latent class analysis", "treatment profile", "treatment trajectory." ], "content": "Introduction\n\nMajor depressive disorder (MDD) has considerable personal and societal consequences, and 15-18% of all individuals will suffer from MDD in their lifetime.1–3 The phenotypic expression characterizes the disorder by a depressed mood, reduced energy and/or loss of interest and enjoyment.4,5 Despite many years of research, the aetiology and pathophysiology of MDD have not been fully clarified.6,7 The disorder is considered heterogenous, reflected by diverse clinical phenotypes, inconsistent biological measures, and assorted and suboptimal responses to treatment.5–10 The available treatments fail to treat all patients successfully; 30% are characterized as treatment-resistant, and even more have a suboptimal treatment outcome.10\n\nGronemann et al. found that the rate of treatment-resistant depression is higher for patients redeeming specific psychotropic co-medicines (e.g. benzodiazepines), suggesting that certain MDD patients have fewer shifts in treatment as comorbid somatic disorders may restrict treatment options due to drug-drug interactions.11 Considering the number of available treatment options, we hypothesize that it is possible to identify clinically meaningful groups of MDD patients based on their psychiatric treatment. Psychiatric MDD treatments include an array of pharmacological and non-pharmacological treatments, and combinations thereof, and is characterized by multiple shifts in treatment over time.12 However, most literature is restricted to studying one treatment at a time instead of treatment profiles. The present study uses a multivariate method (latent class analysis), suitable for identifying data-driven homogenous groups of patients based on a set of information. Here, these groups represent psychiatric treatment profiles based on redeemed prescriptions and non-pharmacological treatments. No studies have previously grouped MDD patients based on their psychiatric treatment by using latent class analysis, nor used psychiatric treatment profiles to investigate treatment trajectories.\n\nThe objectives of this study are 1) to identify treatment profiles based on patients’ psychiatric treatment received during six time intervals positioned before and after depression diagnosis, 2) to link treatment profiles into trajectories over at least three intervals, and 3) to describe patients with similar treatment profiles and trajectories in relation to clinical and sociodemographic characteristics.\n\n\nProtocol\n\nThe present study is a register-based study of patients with a first-time hospital contact for MDD (index date), identified in the Danish National Patient Registry (DNPR) from January 1st, 2011 through December 31st, 2015. Six four-month intervals (Interval 1-6) will be arranged relative to the index date, corresponding to the date of diagnosis; -18 to -14, -4 to 0, 0 to 4, 4 to 8, 18 to 22 and 32 to 36 months (Figure 1).\n\nGrey bars indicate data extraction; the “population data extraction” bar is data necessary to establish the cohort, sub-cohorts and descriptive variables, whereas the “treatment data extraction” bar indicates data required for psychiatric treatment profile (PTP) modelling.\n\nThe study population is a cohort of patients registered at a psychiatric unit, with MDD (International Classification of Diseases 10th revision [ICD-10]): F32 [single episode] or F33 [recurrent]) as a primary diagnosis from 2011 to 2015.\n\nPatients will not be eligible if they 1) had a primary or secondary diagnosis of MDD, dementia (F00-F03) in a period of 15 years prior to index date, schizophrenia (F20), manic episodes (F30) or bipolar depression (F31); 2) if they migrated out of/into Denmark in the same period; 3) if their date of birth was not registered within the Danish Civil Registration System (CRS) for the period 2011-2015; or 4) were younger than 10 years of age at index date (Figure 2).\n\nPatients diagnosed with major depressive disorder (MDD) as primary diagnosis between 01-01-2011 and 31-12-2015 will be included. Patients will be excluded if they A) were diagnosed with MDD, dementia, schizophrenia, manic episodes or bipolar depression 15 years prior to index date, B) migrated in or out of Denmark 15 years prior to index date, C) had incomplete data portfolios or D) were aged under 10 years of age at the time of diagnosis.\n\nInterval-specific sub-cohorts will be established based on a follow-up assessment applied past the index date, i.e. at Interval 3-6. Patients will be excluded if they i) migrated out of/into Denmark throughout an interval, ii) migrated out of Denmark, between two intervals, and did not return before the interval began or iii) died before or within an interval (Figure 3).\n\nEstablishment of the sub-cohort of Interval 5 is used as an example. Patients will be excluded if they i) migrated within an interval, ii) migrated out of Denmark without returning before the beginning of the interval, or iii) died between or within intervals.\n\nThe Danish health care system provides universal coverage and is largely tax-funded.13,14 Data on the Danish population are, mainly for administrative purposes, reported into nationwide registers and cross-linkage at the individual level is enabled by a unique personal identification number.13–15 The CRS dates back to 1968 and includes data on date of birth, sex, vital status, migration, among others.16–18 The DNPR dates back to 1977 and includes data on in- and outpatients from hospitals. Data includes variables regarding diagnostics and treatment.15,17,19,20 The Danish National Prescription Register (NPR) dates back to 1994 and includes data on prescriptions redeemed at community pharmacies by Danish residents.13,21 The National Health Service Register (NHSR) dates back to 1990 and includes data on health care services provided within the primary sector.21,22\n\nPsychiatric treatment\n\nPsychiatric treatment will be defined as at least one treatment redeemed/received by a patient. Data will be retrieved for each interval, and treatments redeemed/received by <100 patients will be excluded.\n\nPharmacological treatment: data on prescriptions redeemed from pharmacies by patients will be extracted from NPR.13 Data to be retrieved are Anatomical Therapeutic Chemical (ATC) codes. The following ATC codes will be retrieved at the 5th ATC level: N03AX09 (lamotrigine), N03AX12 (gabapentin), N03AX16 (pregablin), N05** (psycholeptics), N06A* (antidepressants), N06C* (psycholeptics and psychoanaleptics in combination) and N07B* (drugs used in addictive disorders).\n\nNon-pharmacological treatment: data on treatment received by patients in hospital settings will be extracted from DNPR. Data to be retrieved are SKS codes (Danish Health Care Classification System) at the highest level of specialization, albeit these will be manually clustered after data collection.15 The SKS codes to be retrieved are; BRKP* (all treatments [] with psychoeducation [BRKP]), BRS* (all treatments [] with conversation-based therapy [BRS]), BRT (all treatments [] related to deprivation of liberty and other coercive psychiatric treatments [BRT]) and BRX (all [] other interventions in relation to mental functions [BRX]). Data on treatment received from general and specialized practitioners will be extracted from NHSR, identified as the following SERVICE codes (Danish: SPECIALE koder)22; 24 (all services [] provided by a psychologist [24]), 26 (all services [] provided by children and adolescent psychiatry [26]), 63 (all services [*] provided by a psychiatrist [63]) and 802448 (conversation-based therapy [2448] provided by a general practitioner [80]).\n\nCovariables\n\nVariables for characterization purposes will be: Diagnosis specifications: The ICD-10 code will be retrieved from the psychiatric domain of the DNPR and categorized into F32 (single episode) and F33 (recurrent).4 Additionally, severity will be categorized as mild (F3X.0), moderate (F3X.1), severe (F3X.2 and F3X.3) and unspecified (all other, e.g. F3X.9).4 Sex: Data on sex will be retrieved from the CRS. Age: the date of birth will be received from the CRS and age at index date grouped as; <18 years, 18-34 years, 35-64 years and >64 years. Education: Data on education will be retrieved as the highest completed education at index date and categorized as: group 1 (primary, lower secondary), group 2 (upper secondary, short cycle), group 3 (Bachelor, Master, Doctoral) and group 4 (missing or not classified).\n\nLatent class analysis\n\nPsychiatric treatment profiles will be identified by latent class analysis (LCA). LCA is used to identify latent classes (i.e. groups) of individuals with homogenous characteristics or behavioural patterns, e.g. treatment patterns.23–26 The method will be used to model a finite number of groups at each of the six intervals, and these groups will be referred to as psychiatric treatment profiles.\n\nPatients will be randomly assigned into two datasets to cross-validate the model fitting process.27 Model fitting is an iterative process that fits prespecified model solutions (i.e. the number of groups) to the data. The Bayesian Information Criterion (BIC) will be used to model selection together with a clinical judgement.23,27,28 The fitting process for each dataset will be initiated by a one-class model solution and continued by adding one class to the model solution until the BIC has increased twice.27–29 Once model solutions have been identified for each interval, a comparison of profiles across intervals will take place to assess whether the same profile appears in several intervals. Assignment of patients to profiles will be based on modal assignment, i.e. patients will be assigned to the profile in which they have the highest probability to belong according to the posterior probabilities.24,26 Trajectories will be established by cross-tabulating patients’ profiles at different intervals.\n\nDescriptive statistics\n\nFor characterization purposes, descriptive statistics will be used. Median with interquartile range (IQR) will be used for continuous variables. For categorical variables, frequencies with percentages will be used.\n\nSample size calculation\n\nThe adequate sample size for LCA depends on two main aspects. Firstly, the effectiveness of the indicators to separate classes, and secondly, whether the size of the classes, once separated, is adequate to detect differences.23 Nevertheless, 300 subjects are suggested as a minimum cohort.28 Considering the prevalence of MDD and the eligibility period of five years (2011-2015), we expect that the cohort will exceed 30,000 subjects and that the number of patients of each profile will therefore be adequate.\n\nStatistical software\n\nThe forthcoming study will access data from research servers at Statistics Denmark.13 Statistical Analysis System (SAS) Enterprise Guide® (SCR_008567) will be available on the research servers from which data management will be led. Alternatively, the open-source software R-Studio (SCR_001905) can be used.\n\n\nDeliverable/data synthesis\n\nIn the research article, the results will be presented in graphs, tables and diagrams. Two different graphs will be computed; plot constructs with BIC (y-axis) versus model solutions (x-axis), and plot constructs with the conditional probability (y-axis) versus psychiatric treatment (x-axis).27 These graphs will represent the model fitting and model selection process, respectively. The identified profiles, their prevalence and treatment patterns (conditional probabilities) will be presented in a table alongside profile-specific patient characteristics (Table 1). Likewise, the most frequent trajectories will be represented in a table (Table 2). Lastly, the most frequent trajectories will be depicted by a Sankey diagram in order to visualize the shifts between profiles and the magnitude of these shifts.\n\n Indicate where results will be presented.\n\nMDD: major depressive disorder. Int.: interval. IQR: interquartile range.\n\n* Indicate where results will be presented.\n\nMDD: major depressive disorder. IQR: interquartile range. NA: not applicable.\n\nThe register-based study design does not require approval from an ethics committee according to Danish law.21 The Danish Data protection Agency has approved the study (reference number P-2020-88).\n\nThe results will be submitted for publication to an international peer-reviewed journal.\n\n\nDiscussion\n\nTime intervals are positioned relative to patients’ index dates, thus intervals are patient-specific, meaning that the same interval is likely to occur at different time points for different patients and thereby be influenced by time-specific trends in treatment, legislation, guidelines, or new treatments. However, as the inclusion period only spans five years, we do not expect this to have a major impact on the results. Psychiatric treatment profiles are based on treatments redeemed/retrieved within a four-month interval. Consequent, any shifts/discontinuation of treatment that may occur within these four months will not be represented by the psychiatric treatment profiles. Thus, the profiles could, misleadingly, be interpreted as polypharmacy or false treatment combinations. However, as many treatments are prescribed for three months at a time, we do not expect this to be an issue. In addition, we expect fewer treatment shifts in intervals further away from index time.\n\nThe ATC codes will be assessed at the 5th level of the ATC system. At this level, the number of indicator variables (of the pharmacological treatment) is maximized. This might increase the complexity and limit the power of the information gained from the psychiatric treatment profiles. Nevertheless, there are clinical differences at the 5th level of the ATC system (e.g. adverse effects), and an assessment at the 4th level of the ATC system would mask clinical preferences.30\n\nPsychiatric treatment that are redeemed/received by less than 100 patients will be excluded, i.e. treatment rarely redeemed/received by patients will not be included. This will lead treatment given to a minor subset of the study population to go undetected.\n\nThe universal coverage in Denmark minimizes the risk of selection bias.15 The overall validity of a single depression diagnosis (F32.0-3) identified in DNPR was found to be 72.8% for children and adolescents.31 The overall validity of the same diagnoses was found to be similar for adults (overall validity of 75.4%).31,32 We expect the validity to be similar for a recurrent depression diagnosis (F33.0-3). The eligibility criteria will influence the study population composition which may introduce false homogeneity, e.g., the criterion of not having certain psychiatric diagnoses 15 years prior to index date can exclude patients with psychiatric comorbidity. This may skew the representativeness of the study population and thereby limit generalizability of the results.\n\nThe mandatory and mainly electronic reporting of data into registers minimizes the overall risk of information bias.13,15 Pharmacological treatment data will exclusively be collected from the NPR. The register is considered complete and valid but still considered to have some drawbacks.13 The NPR lacks data on the indication of use, treatment duration, dosage, and adherence. The study, therefore, assumes that the pharmacological treatment is intended for MDD and consumed by the patient.13 Moreover, the NPR does not include pharmacological treatment received at hospitals; however, less than 1% of all antidepressants are given at the hospital and the majority of patients are treated by their general practitioner outside hospital settings.33,34 We do therefore not believe that lack of data on pharmacological treatment at hospitals will influence our results.\n\nNon-pharmacological treatment data will be extracted from the DNPR and NHSR. Change in registration codes over time, varying registration practice and the lack of mutual exclusiveness can introduce information bias in data from the DNPR.15 We believe that the same concerns are likely to influence data from the NHSR. Furthermore, the NHSR does not include treatment not covered by the Danish health care insurance (Danish: Sygesikringen), therefore data on patient-financed treatment is not included.\n\nAll data management will be internally validated.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nRibeiro Â, Ribeiro JP, von Doellinger O : Depression and psychodynamic psychotherapy. Braz. J. Psychiatry. 2018; 40(1): 105–109. PubMed Abstract \| Publisher Full Text\n\nThe Danish Health Authority: The Burden of Disease in Denmark [Document].2015.\n\nKessler RC, Bromet EJ: The epidemiology of depression across cultures. Annu. Rev. Public Health. 2013; 34: 119–138. PubMed Abstract \| Publisher Full Text\n\nThe World Health Organization: The ICD-10 Classification of Mental and Behavioural Disorders: Clinical descriptions and diagnostic guidelines [Document].\n\nThe Danish Health Authority: Reference program of unipolar depression in adults [Document].2007.\n\nDean J, Keshavan M: The neurobiology of depression: An integrated view. Asian J. Psychiatr. 2017 Jun; 27: 101–111. PubMed Abstract \| Publisher Full Text\n\nKennis M, Gerritsen L, van Dalen M , et al.: Prospective biomarkers of major depressive disorder: a systematic review and meta-analysis. Mol. Psychiatry. 2020; 25(2): 321–338. PubMed Abstract \| Publisher Full Text\n\nThe Danish Health Authority: Professional Guidelines for Psychologist Referral [Document].2012.\n\nMenke A: Is the HPA Axis as Target for Depression Outdated, or Is There a New Hope?. Front. Psych. 2019 Feb 10; 10: 101. PubMed Abstract \| Publisher Full Text\n\nManchia M, Pisanu C, Squassina A, et al.: Challenges and Future Prospects of Precision Medicine in Psychiatry. Pharmacogenomics Pers. Med. 2020; 13: 127–140. PubMed Abstract \| Publisher Full Text\n\nGronemann FH, Jorgensen MB, Nordentoft M, et al.: Socio-demographic and clinical risk factors of treatment-resistant depression: A Danish population-based cohort study. J. Affect. Disord. 2020 Jan 15; 261: 221–229. PubMed Abstract \| Publisher Full Text\n\nKeks N, Hope J, Keogh S: Switching and stopping antidepressants. Aust. Prescr. 2016 Jun; 39(3): 76–83. PubMed Abstract \| Publisher Full Text\n\nPottegård A, Schmidt SAJ, Wallach-Kildemoes H, et al.: Data Resource Profile: The Danish National Prescription Registry. Int. J. Epidemiol. 2016 Oct 27; 46: dyw213–dy798f. PubMed Abstract \| Publisher Full Text\n\nSchmidt M, Schmidt SAJ, Adelborg K, et al.: The Danish health care system and epidemiological research: from health care contacts to database records. CLEP. 2019 Jul; 11: 563–591. PubMed Abstract \| Publisher Full Text\n\nSchmidt M, Schmidt SAJ, Sandegaard JL, et al.: The Danish National Patient Registry: a review of content, data quality, and research potential. CLEP. 2015 Nov; 449. Publisher Full Text\n\nSchmidt M, Pedersen L, Sørensen HT: The Danish Civil Registration System as a tool in epidemiology. Eur. J. Epidemiol. 2014 Aug; 29(8): 541–549. PubMed Abstract \| Publisher Full Text\n\nErlangsen A, Fedyszyn I: Danish nationwide registers for public health and health-related research. Scand. J. Public Health. 2015 Jun; 43(4): 333–339. PubMed Abstract \| Publisher Full Text\n\nPedersen CB: The Danish Civil Registration System. Scand. J. Public Health. 2011 Jul 1; 39(7_suppl): 22–25. Publisher Full Text\n\nLynge E, Sandegaard JL, Rebolj M: The Danish National Patient Register. Scand. J. Public Health. 2011 Jul 1; 39(7_suppl): 30–33. Publisher Full Text\n\nMors O, Perto GP, Mortensen PB: The Danish Psychiatric Central Research Register. Scand. J. Public Health. 2011 Jul; 39(7_suppl): 54–57. Publisher Full Text\n\nThygesen LC, Daasnes C, Thaulow I, et al.: Introduction to Danish (nationwide) registers on health and social issues: Structure, access, legislation, and archiving. Scand. J. Public Health. 2011 Jul 1; 39(7_suppl): 12–16. PubMed Abstract \| Publisher Full Text\n\nSahl Andersen J, De Fine ON, Krasnik A: The Danish National Health Service Register. Scand. J. Public Health. 2011 Jul 1; 39(7_suppl): 34–37. Publisher Full Text\n\nSinha P, Calfee CS, Delucchi KL: Practitioner’s Guide to Latent Class Analysis: Methodological Considerations and Common Pitfalls. Crit. Care Med. 2021 Jan; 49(1): e63–e79. PubMed Abstract \| Publisher Full Text\n\nLanza ST, Cooper BR: Latent Class Analysis for Developmental Research. Child Dev. Perspect. 2016 Mar; 10(1): 59–64. PubMed Abstract \| Publisher Full Text\n\nCollins LM, Lanza ST: “General Introduction”. Latent Class and Latent Transition Analysis: With Applications in the Social, Behavioral, and Health Sciences. 1st ed.John Wiley & Sons, Inc.; 2010; 3–22.\n\nKongsted A, Nielsen AM: Latent Class Analysis in health research. J. Physiother. 2017 Jan 1; 63(1): 55–58. Publisher Full Text\n\nCollins LM, Lanza ST: Parameters estimation and model selection. Latent Class and Latent Transition Analysis: With Applications in the Social, Behavioral, and Health Sciences. 1st ed.John Wiley & Sons, Inc.; 2010. 77–110.\n\nWeller BE, Bowen NK, Faubert SJ: Latent Class Analysis: A Guide to Best Practice. J. Black Psychol. 2020 May 1; 46(4): 287–311. Publisher Full Text\n\nNylund KL, Asparouhov T, Muthén BO: Deciding on the Number of Classes in Latent Class Analysis and Growth Mixture Modeling: A Monte Carlo Simulation Study. Struct. Equ. Model. Multidiscip. J. 2007 Oct 23; 14(4): 535–569. Publisher Full Text\n\nGerald G, Kylie T, Hill S, et al.: How Should Primary Care Doctors Select Which Antidepressants to Administer?. Curr. Psychiatry Rep. 2012 Aug; 14(4): 360–369. Publisher Full Text\n\nFrederiksen LH, Bilenberg N, Andersen L, et al.: The validity of child and adolescent depression diagnoses in the Danish psychiatric central research register. Acta Psychiatr. Scand. 2021; 143(3): 264–274. PubMed Abstract \| Publisher Full Text\n\nBock C, Bukh JD, Vinberg M, et al.: Validity of the diagnosis of a single depressive episode in a case register. Clin. Pract. Epidemiol. Ment. Health. 2009 Feb; 5(5): 4. Publisher Full Text\n\nCouncil for the Use of Expensive Hospital medicines: Pharmacological treatment of unipolar depression in adults [Document].2015.\n\nThe Danish Health Authority: National Clinical Guideline for the Non-pharmacological treatment of Unipolar Depression [Document].2016." }	[ { "id": "186853", "date": "14 Sep 2023", "name": "Esteve Gudayol-Ferré", "expertise": [ "Reviewer Expertise Neuroimaging FMRI", "neuropsychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work entitled \"Profiles and trajectories of treatment around the diagnosis of major depressive disorder: a research protocol for a Dane register-based study\" is interesting. Overall, it is well planned, and its important details have been taken into account. The text and the figures are well-developed, and the protocol, in general, is clear and complete. For this reason, I only have some comments that can contribute to improving some aspects of the protocol.\nThe paragraph about the sub-optimal treatment outcomes of depression could be developed with a little more depth, given its importance for the rationale of the present protocol.\nThe authors could also explain why they chose the time intervals to measure antidepressant outcomes and why this particular number of outcome points. The rationale for the specific time window to collect the data must be briefly explained. Why collect specifically the register of patients between 2011 and 2015?\nThe authors will select participants as young as ten years old in their study. Have you considered selecting only adult participants? Why do they also choose children? Will the authors carry out data analysis separating children and adults by age group?\nI know it would be a significant added difficulty for the proposed data analysis. Still, the authors have not considered including in the analysis some medical comorbidities that are frequently associated with depression or have a high incidence and prevalence in their population. ? Alternatively, consider excluding patients with such conditions from your study. Or, at the very least, plan a priori data analysis by subgroups of patients: one with the entire sample. Another only with the participants that constitute a population \"clean,\" that is to say, with only MDD...Whether the authors choose one option or the other, they must try to control this important variable. Undoubtedly, it is my major concern about the present protocol. And the point that is most important to resolve before starting the study.\nFrom a theoretical point of view, it is questionable that all kinds of deprivation of liberty can be considered a treatment for depression. Do the authors mean forceful hospitalizations? The authors must define in specific operational terms this variable.\nMuch more important, the \"conversation-based therapy\" variable is not well defined either. Some psychotherapies have amply proven to be an effective treatment for depression, but not all of them. It would be convenient to keep only those interventions of proven efficacy if the authors intend to define a variable that groups them all together.\nI am not an expert in data-driven data analysis or machine learning. That's why I have no suggestions about this section.\nI believe that the discussion contains an adequate treatment of the possible limitations of the study, and I have no comments on this section.\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": [] } ]	1	https://f1000research.com/articles/11-193
https://f1000research.com/articles/11-192/v1	15 Feb 22	{ "type": "Study Protocol", "title": "A formative usability study of workflow management systems in label-free digital pathology", "authors": [ "Markus Jelonek", "Arne Peter Raulf", "Eileen Fiala", "Joshua Butke", "Thomas Herrmann", "Axel Mosig", "Markus Jelonek", "Arne Peter Raulf", "Eileen Fiala", "Joshua Butke", "Thomas Herrmann" ], "abstract": "This research present a formative usability study that investigates the usability of different workflow management systems in the field of biomedical data analysis. Specifically, a task in the field of so-called label-free digital pathology has been analysed and one graphical user interface based workflow and one script-based workflow to solve the task was investigated. The main intention is to gain first in-sights into the systematic study of usability in the context of biomedical image analysis, and formulate experiences and guidelines for future usability studies dealing with workflow management systems. Embedded in a specific setup dealing with label-free digital pathology, the core question behind this research is how usability studies for scientific workflow management can be conducted, and how they can be used systematically to improve such tools. Further, this study address specific questions about the resource utilisation and management of usability studies, including the recruitment of participants as well as the design of specific workflows to be investigated.", "keywords": [ "Scientific Workflow Management", "Bioimage Analyis", "Usability Study" ], "content": "Introduction\n\nIn recent years, numerous workflow management systems (WMSs) for the reproducible analysis of high-throughput experimental data in life science research have been developed.1–5 It is well-established that such WMSs are indispensable to warrant transparency, reproducibility and interoperability of increasingly complex data analysis tasks that typically combine a number of different data analysis tools and subtasks. Matching the needs of different user groups and application settings, WMSs have been implemented as either graphical user interfaces1–4 or as script-based tools,5 addressing a broad variety of user groups and an equally broad range of needs for user interaction or operability on large data sets.\n\nMany of the prominent WMSs have been developed and maintained over many years and are now mature software systems whose relevance and popularity are undermined by large citation numbers. However, apparently little attention has been spent on systematically and formally exploring workflow management from the viewpoint of usability. Yet, usability is of interest in many respects. First, a detailed investigation of software usability can guide future developments of specific tools, and more generally may help to summarize experiences to formulate guidelines for future developments.6,7 Second, the ergonomics of workflows affect the outcome of data-centric research studies. Since many studies and corresponding data analysis workflows involve a human-in-the-loop,8 usability aspects inevitably determine the inductive bias of obtaining insights from data. A thorough investigation of usability is thus not just a matter of assessing convenience and efficiency, but also a matter of elucidating the role of software in the scientific method.\n\nIn this contribution, the results of a formative usability study in the context of label-free digital pathology9 is presented. The goal of this study is to provide a first case study on how usability studies for scientific workflow management can be conducted, and how they can systematically be used for improving WSMs. Specifically, a study that investigates and compares two distinct groups of users following two different workflows was conducted to solve one and the same image analysis task.\n\nThis study deals with a setting in label-free digital pathology based on hyperspectral infrared microscopic imaging data and it is based on a workflow originally established by Kallenbach-Thieltges et al. 2013,9 which was the basis for a number of applications of infrared microscopy in different clinical settings.10–12 In label-free digital pathology, tissue samples are measured using an infrared microscope. The infrared microscope yields a hyperspectral microscopic image with a spatial resolution of around 5 μm, where each pixel is represented by an infrared spectrum covering a wavelength bandwidth of several hundred optical channels. To identify disease-associated tumor regions in tissue samples, the images need to be pre-segmented using unsupervised learning approaches which allows to extract training data for a supervised classifier, as illustrated in Figure 1.\n\nPanel A) shows the workflow as abstract scheme, while panel B) shows the same workflow implemented in the Orange framework. Panel C) gives an impression of the main implementing this workflow in OpenVibSpec.\n\nThis study involves two different implementations of this workflow. First, a script-based implementation from the OpenVibSpec tools was used,13 which constitutes a Python re-implementation of the workflow used in previous studies.9–12 The second implementation was based on the graphical user interface (GUI) of the Orange WMS.1 Furthermore, two distinct user groups with different levels of programming skills and life science background were recruited to investigate their usage patterns across the two different implementations. In addition, this study addresses the question of how to evaluate a given workflow for biomedical image analysis.\n\n\nUsability testing\n\nAs documented by the International Standards Organisations (ISO) in ISO 9241, the usability of a product or a system is defined as the extent to which a user can use the system to achieve his/her goals “effectively, efficiently and with satisfaction”.14 In its core, usability is not one single factor of a system or product but multiple factors of the system or product contribute to or reduce its usability. These factors or concepts are: learnability, efficiency, memorability, errors and satisfaction.6 Usability is seen as relevant during the whole lifetime of a product or system, from development to procurement, and is an important aspect of the perceived user experience (UX) of a product or system.\n\nTo evaluate the usability of a system, a variety of methods and types of usability tests or inspections can be used. Such evaluations can differ, for example, in the participants’ knowledge (expert vs. novice user), in its test design (task-based test vs. cognitive walkthrough), or if it is done during the development or with a final version of the system (formative vs. summative evaluation).6,15,16 Besides test-based evaluations, heuristic usability inspections are also commonly used.17\n\nFormative studies are of a rather exploratory nature and can be conducted very early in the development phase of systems, even on the basis of simple wireframes or prototypes to define requirements and reflect on the interaction design at an early design stage.16 In these studies, mainly qualitative data is collected, for example comments and impressions by users regarding the interaction design of the system, if and how successful a task could be completed or how they perceived the systems feedback (cf. Ref. 15). Common methods for formative usability studies are, for instance, thinking aloud tests, in which users are encouraged to articulate their thoughts during use or task-based tests with follow-up interviews. In summative studies, a more formal approach is taken and attempts are made to demonstrate the effectiveness of a newly developed system. To conduct a summative usability test, at least functioning prototypes are needed. A typical goal of a summative test would be to highlight the benefits of a new system or interface to other existing ones. However, the boundaries of formative and summative testing are not rigid, as a summative test could also be applied after several development stages of a system.\n\nTo assess the usability of a system, a combination of different metrics and methods can be applied to collect the data of interest. However, the type of test can affect the number of participants in a study. For example, an expert-based usability evaluation can possibly be applied with a smaller number of participants than for example user-based usability tests. Yet, the number of needed participants in usability tests is under dispute.18–22\n\nIn this study, a formative approach for the usability evaluation was used. The primary aim of this study is not to compare the efficiency of both tools but rather to explore possibilities and constraints designing usability studies for scientific workflow management tools and how such usability studies contribute to their improvement.\n\n\nMethods\n\nIn order to generate a broad spectrum of insights and perspectives, a formative approach was chosen for the usability evaluation of the two WMS. A task-based study with participants was conducted, in which participants were asked to use a combination of supervised and unsupervised machine learning algorithms to classify microscopic data of tissue images, as shown in Figure 1. During use, observations were noted by the study facilitator and participants were asked to express thoughts aloud. After completing the tasks, a semi-structured interview was conducted with each participant to verify the observations made and to solicit further perspectives. The interview incorporated questions based on Nielsen’s usability heuristics.17\n\nTwo alternative implementations of the workflow were investigated, one that could be used via script and one that offered a GUI. For the script-based implementation of the workflow, the Python-based implementation provided in the OpenVibSpec tools13 was utilized with a Jupyter notebook23 as an interface (See source data).24 The implementation provides structured access to perform clustering of image spectra, to extract training spectra from annotation masks, and to train and validate supervised classifiers. Following common practice,9 k-means clustering25 was employed for unsupervised segmentation combined with random forests26 for supervised classification.\n\nThe study GUI based implementation relies on the functions provided by the Orange framework1 (See source data).24 In Orange, a workflow can be created by connecting different widgets. These widgets represent certain data operations on a data set and usually allow some kind of configuration. As illustrated in Figure 1, the data for the workflow was pre-established in the interactive Orange GUI, as data import and export operations were already added. Thus, users were only required to create the workflow with suitable data operations and adjust parameters. This adaptation of the workflow saved considerable time for the study, as the import and export operations demanded a noticeable amount of time. The final workflow is illustrated in Figure 1A.\n\nIn order to facilitate at least basic comparison between the two implementations, this study was limited to those methods that are implemented in both platforms, noting that both Orange and OpenVibSpec are based on the same standard libraries, most notably Scikit-learn27 and Numpy.28\n\nFor both implementations, participants were briefed with a targeted training video to explain the technical realization of the workflow, in order to warrant the necessary level of prior knowledge for the study (See source data).24 Participants had the chance to watch the video or parts of the video again at any time during the study.\n\nThe data set was derived from previously published studies investigating colorectal carcinoma using infrared microscopy.11,29 In brief, the complete data set comprises infrared microscopic images of roughly 200 tissue-microarray (TMA) spots of more than 100 different patients, where each spot is roughly 1 mm in diameter and represented by an infrared microscopic image covering roughly 250×250 pixel spectra. TMA slides were purchased from US Biomax Inc., MD, USA, and the infrared microscopic images are accompanied by conventional hematoxylin and eosin (H&E) stained images which were acquired subsequent to infrared microscopy. For the script-based implementations, many images of a complete TMA spot were used as a basis for the study. For the GUI-based implementation, images were reduced in size to 224×224 pixels for the training data and 195×210 for the test data in order to meet constraints in terms of computational resources and time window per participant.\n\nFor the study, 22 participants (7 females, 15 males) were recruited in total, among students of two different study programs at the Ruhr-University Bochum with ages ranging from 23 to 39 years (M = 26.05, SD = 3.7) (see Underlying data30).\n\nIn order to take account of the study’s objectives in the selection of test users, two target groups were deliberately sought which correspond to the users of the tools compared, namely Orange and OpenVibSpec.\n\nThe first group was recruited among students with at least advanced undergraduate knowledge in a life science study program and no or limited programming skills (6 females, 5 males, mean age = 25.27, SD = 2.83). The second group was recruited among computer science students at comparable level, possessing good programming skills, but no or limited background in the life sciences (1 female, 10 males, mean age = 26.82, SD = 4.26).\n\nTo counteract a bias of the study by specifically selected test persons, all participants were deliberately selected in such a way that they did not have any special expertise in the actual question being compared, i.e. microspectroscopic tissue characterisation. Based on this, all participants were provided with the same training material, which should shed more light on the background of the data origin and the methods used. Care was taken to ensure a lightweight introduction was given to enable all participants to concentrate fully on the task during the actual test series.\n\nThe implementations were made available to participants on a desktop computer equipped with an Intel i7-2600 CPU at 3.40Ghz and 8 GBytes memory. The actual implementations were installed on a remote Linux server with 64 CPUs and 512 GBytes of main memory, and made available on the desktop computer through a remote desktop connection, which was also equipped with a Linux OS.\n\nThe task had to be adapted in such a way that it could be carried out with both tools, but at the same time produce a result that could be interpreted in the sense of the introductory material provided to the participants; This last requirement and the general orientation of the two tools caused a discrepancy in the execution speed of the Orange tool, which is describe in more detail in the results section. Since the data reading and saving processes also took several minutes, they were skipped in the task for Orange and the participants were given a minimally prepared workflow in which the training and test data widgets were already provided.\n\nThe procedure for each participant was conducted as follows: First, each participant completed a demographic questionnaire and gave their consent to participate in this study (see Underlying data30). Then, participants watched an introduction video with educational material on workflow-based hyperspectral infrared microscopic imaging data analysis. After watching the video, participants started the task with either the GUI tool or the script-based implementation. The instructions to complete the script-based workflow were given in the Jupyter environment.31 For the GUI workflow, participants received the instructions on how to complete the task on paper and PDF. During the study, participants had the chance to ask questions, e.g. if they got stuck or had any other kind of problem to complete the task. While using the tools, the participants were also asked to think out loud. During the task, their screen was recorded and notable usability issues were documented by the study facilitator. After completing both workflows, a semi-structured interview was conducted with each participant in which the usability of both tools was focused.\n\nThis semi-structured interview consisted of general questions about the participants and their prior knowledge, as well as questions integrating Nielsen’s usability heuristics17 (see Underlying data30). The interviews made it possible to specifically question observations that we had documented with the respective participants and to judge their comments and wishes more accurately.\n\n\nResults\n\nThe results are structured into three subsections. First, is the specific task-related observations and results gained from participants’ use of both tools. In the following several usability problems are provided with suggestions for improvement. All the usability issues that occurred were not listed, however the examples that had a major negative impact on task completion in this study were mentioned. Lastly, task completion and task understanding in each of the two participant groups were examined, and how user studies on real data can be conducted to help make WMS available to a wider range of users was analysed.\n\nTask completion\n\nRegarding the task completion time, the group of life science students needed slightly longer to finish the task in OpenVibSpec and, on average, around 5 min longer to finish the task in Orange (see Table 1) (see Underlying data).30 The task completion time did not differ notably between Orange and OpenVibSpec for the computer scientists. The time differences for the life science group were on average around 3 min. These results suggest two aspects. First, the task duration was reasonably evenly distributed between the two systems, as both groups needed around the same time to finish the task. Second, the results also show that the computer scientists finished the task faster with both tools in this study. It should be noted, however, that participants were not asked to finish the task as quickly as possible, but could do it on their own pace. Therefore, the faster completion time of the computer scientist group can probably be justified by a more practiced use of computers in general.\n\n* n=10, as one participant could not finish the task with Orange due to technical problems.\n\nTask complexity\n\nThe task was rated as quite complex by the life scientist group. Using the scripted approach with OpenVibSpec, all participants managed to complete the task successfully. However, 10 out of 11 participants of the life sciences group needed very close supervision and a fair amount of assistance, as the learning curve seemed too steep without prior programming knowledge. For example, exchanging a simple placeholder variable with a concrete integer value for the segmentation of the image was not obvious to many, although it was clearly described in the instructions. Without the intervention of the study facilitator, some participants would have already given up on this task at this point. Questioned afterwards, the participants also stated that the instructions actually clearly described the exchange of the placeholder variable for an integer value. Some said they were simply overwhelmed by the task and by the code. As for the computer science group, still 5 out of 11 participants needed some sort of support during the script-based task. Overall, interviews indicated that the task was easier to solve with the GUI. Especially participants who found the script-based variant somewhat discouraging positively emphasized the GUI, as the GUI enabled them to follow the workflow at least visually and, despite the instructions, they had the feeling of being in control. However, even with step-by-step instructions for the GUI, it was a highly challenging task for novice users as most of them relied on support by the study facilitator at one point or the other. In conclusion, task complexity can be interpreted in several ways. The different perception of the two approaches may have been due to the differently prepared instructions (once text instructions in Jupyter, once screenshots for Orange). However, this is contradicted by the fact that at least one participant in the Life Science Group was able to solve the task who had neither Python nor programming experience. Instead, the perception of complexity seems to be based more on programming skills in general. Many participants, especially in the life science group, had to learn how to run programming code in the Jupyter environment at the beginning of the task. Some participants even had difficulties recognizing which sections belonged to the instructions and which belonged to the script on their own, until they received support from the study facilitator. Such difficulties made the barrier to solve the task even higher for these participants.\n\nComprehension of results\n\nParticipants’ understanding of the workflow outcomes was quite heterogeneous. Although all participants were given a basic introductory video to watch before the first task, four life scientists and four computer scientists were unable to give any real conclusion about the outcome images, such as what the classification of the images could mean and how one would proceed further. In total, four participants (two Computer Scientists, six Life Scientists) expressed that they had followed the steps of the instructions but could not elaborate on the context of the subject matter. Overall, it had been easier to understand the workflow in Orange, since the widget names would have at least made it understandable that a data set was manipulated with several operations. The majority of participants (nine computer scientists, five life scientists) were able to explain that the workflow applied artificial intelligence methods to classify tissue data. Furthermore, they were also aware that they could not interpret the results in more detail and would have to present the result to an expert to repeat the clusterings with other parameters if necessary. This shows that although the participants did not know the dataset before participating in the study, nor had they used the tools before, they were able to perform the task and also were able to comprehend it well in terms of its subject matter. In addition, one participant explicitly pointed out that the resulting images lacked a unique assignment of index colors. It was impossible to understand how the method decided which areas of the image were clustered together. Here, the participant mentioned the use of explanatory labels to understand the clustering.\n\nSevere usability issues\n\nThe usability issues presented here refer primarily to those that made designing the task for the study challenging and those that hindered or negatively impacted participants’ task completion.\n\nLearnability\n\nAs mentioned in the previous section, participants of the life science group had severe problems in completing the task on their own with the script-based approach of OpenVibSpec. The learning curve seemed too steep for the majority of these participants despite detailed instructions. However, the learnability of OpenVibSpec was disrupted in that many participants first had to learn how to use the Jupyter notebook, e.g. to run scripts or even to understand the controls at all. The task became considerably easier if someone had previous experience in programming. Participants with prior experience in Python and Jupyter notebooks had little to no problems focusing on and solving the task. In comparison, all participants were able to replicate the workflow independently with the GUI of Orange, even though none of the participants knew the application beforehand.\n\nEfficiency\n\nPrior to the usability test, during the preparation of the study, longer computing times for Orange was recognized. Therefore, the data set used in this study was reduced for Orange. Since the data reading and saving processes also took noticeably longer computing times, they were skipped in the task for Orange, and the participants were given a minimally prepared workflow beforehand. During the interviews, several participants from both groups presumed that the tools share the same code base, except that one offers a GUI and the other only script-based. These assumptions made by the participants suggest that the changes to the workflow (smaller data set for Orange) and to the task (skipping the import and export operations in Orange) resulted in both tools being considered equivalent in terms of efficiency and considered comparable.\n\nIn addition, it can be noted that participants who had no programming experience rated the efficiency of Orange better. The reason given was learnability: In OpenVibSpec, a longer training period would be necessary and programming experience would be required, whereas Orange could be operated directly via the GUI.\n\nError messages and error prevention\n\nAn important usability criterion is the handling of error messages, which should represent the error in a human-readable form. During the study, error messages occurred in both tools that were basically Python error messages and could only be interpreted by participants with programming experience. For all other participants, the study facilitator had to intervene and provide assistance to the participants. In addition to this, in the Orange GUI it was not always clear to the participants when an error message was displayed and when it was only a warning message. The occurrence of such error and warning messages led to uncertainty in many participants’ completion of the task, which is why the study facilitator explained the messages shortly and participants could continue with the task.\n\nAnother usability issue concerns error prevention in general. An example for a missing error prevention that hindered participants to achieve the expected resulting image occurred during the classification with the random forest algorithm in Orange. Only 7 out of 11 participants from the computer science group were able to successfully complete the task. Three achieved a different result image via incorrectly connected widgets. In the life sciences group, 6 out of 11 participants were able to correctly solve the task with Orange. For 5 out of 11, the same connection error of two widgets occurred. This error was a result of a rather simple usability issue while creating the workflow. However, users did not realize they had done something wrong until they saw the resulting image, as the feedback given by the GUI was not explicit and clear enough. These participants did not notice that the order in which they connected the training and test data to a specific widget led to an automatic classification of the data by the tool as training or test data. Due to the incorrect order of the connections, training data was labeled as test data and vice versa (see Figure 2). Here, the feedback to the users was not clear enough, as it was not noticed by those to whom it happened. On the other hand, this error could have been prevented if the order of the connection had not mattered. In OpenVibSpec such an error did not occur because an automatic interpretation of the dataset is not offered, but one had to explicitly specify the training dataset or test dataset in the method call.\n\nIf afterwards the the training data (‘Train’) was connected with ‘Test and Score’, the tool labeled the training data as test data. This issue occurred to 8 participants in total.\n\nThe data revealed that the participation of two different user groups resulted in a broader range of views, task-related observations, and usability problems found.\n\nFor example, according to the statements in the interview and the observations during the study, it was apparent that most participants had a more pleasant user experience with the GUI tool, in particular in the life scientists group. Although 6 out of 11 participants in this group had difficulties understanding the workflow and the results of the workflow, the GUI gave them at least the possibility to try to comprehend the data operations and the feeling to be in control whereas the script-based approach seemed too complex to be comprehended for many. Participants of both groups believed that as non-programmers they would prefer the GUI-tool, as the script-based approach with OpenVibSpec would require too much prior knowledge. Instead, the GUI has used familiar interaction techniques, for example, using drag-and-drop or context menus.\n\nWith regard to the exploratory behavior of the participants, no real differences were found between both groups. However, in the life science group there were three participants who performed the same k-Means clustering three times in OpenVibSpec, while in the computer science group there was only one participant who repeated the clustering with the same cluster size three times. All other participants took a more exploratory approach to the task and chose three different cluster sizes (Table 2). In addition, there were also two participants in the life science group who chose a clustering of c=1, which ultimately resulted in a monochromatic image, suggesting that they were not entirely sure what the clustering was doing, at least beforehand. In Orange, no participant attempted exploratory testing of cluster sizes. Instead, the k-means widget was used without modifying the settings, resulting in every participant using the same cluster size (c=5). To get to the settings for the widget, one would have had to open it. However, this was neither requested nor suggested in the task description for Orange. Participants would probably have noticed the cluster size if it had been visually reflected in the widget and perhaps could have been changed directly in the GUI without having to open the settings. Such visual feedback directly visible workflow could promote exploration of different clusterings. However, due to the two-step procedure (first open the widget settings, then change the cluster size), the participants neither knew nor questioned the number of clusters used in the workflow in Orange.\n\nAnother difference between the groups was that some participants in the life science group were negative about the script-based approach. During task processing with OpenVibSpec, there were at least two participants who clearly verbalized their frustration with the tool, especially when errors occurred. Another participant expressed a strong preference not to have to go through the workflow, but expected the tool to simply analyze the image data and provide him with the results without having to start multiple clusterings or algorithms, so that he would hardly have to interact with the tool at all.\n\nRegarding the perceived efficiency, six participants (four life scientists and two computer scientists) rated Orange as more efficient than OpenVibSpec and two participants (one of each group) rated the efficiency of both tools as equal, although the computing times of Orange were noticeably longer. One possible explanation for this perception is that in OpenVibSpec three runs for k-means were prepared and the majority of participants explored differences between cluster sizes (see Table 2). In addition, the calculations lasted longer when participants used a higher cluster size. Another possible explanation for the perceived efficiency is, that participants (especially from the life science group) felt more comfortable using the graphical widgets.\n\n\nDiscussion\n\nAs planned, the study revealed many usability problems in either of the tools and on different levels of severity. Several issues were discovered as a result of inviting two different user groups in the study. For example, the high barrier entry for non-programmers and script-based workflow management system was particularly evident for the life science group. Here we could show that even the simplest changes in the scripts were challenging for novice users and could only be solved with assistance of the study facilitator. Accordingly, training or tutorials would be necessary for users if they are to apply such script-based tools for their purposes. In addition, some usability problems (including the long loading times for Orange) could already be noticed during the preparation of the study. Ideally, these problems should have been fixed prior to conducting the study, so that the data import could have been performed in both tools.\n\nAnother challenge that frequently occurred was that participants had difficulty understanding or interpreting the results. The problem of interpretability could not be traced back exclusively to one principle in the approach selected, but it is particularly likely that either more feedback needs to be worked out based on the methods used. However, it is also possible that the question of bioimaging needs to be more linked to the workflow used rather than the tool.\n\nConsidering the amount of effort for the study and the usability problems found, it seems likely that a smaller number of participants would have been sufficient for the results. This illustrates the problem of the number of participants to be used quite well (see Section 0). Probably, even with small-step user tests, severe usability problems would have already been noticed. In part, we have already encountered this during the development of the tasks for the study (e.g., efficiency problems). However, such studies reveal numerous usability issues and requirements for changes, as well as many views and ideas to be interpreted, which, if implemented well, can ultimately lead to better tools and larger user bases.\n\nIn order to clarify the importance and benefit of usability tests in the field of WMSs, this study’s methodical approach requires reflection and discussion. One challenge was to design a task that covered roughly the same aspects in two different tools (GUI vs. script). To achieve this,several more modules/plugins had to be integrated into Orange that were not included in the main application. This in turn, only became apparent with the need to create comparative tasks in both tools. The development of the tasks for evaluation therefore already revealed certain issues (e.g. missing modules, efficiency problems). Such usability issues could probably have been found systematically for both tools with a heuristic analysis before the study. Thus, depending on the scope of the analysis and available resources, even a heuristic inspection of the tools could find many problems and opportunities for improvement before conducting a larger scaled user study.\n\nDue to the participation of novice users and the exploratory nature of the study, the results are limited with respect to the use of bioimage processing. Here, in comparison, it would be interesting to see how experienced users working in the field would handle the two tools. In particular, an exploratory strategy, including various clusterings, and the interpretation of results could vary with experienced users. However, a positive aspect of our approach was that different user groups were selected for the study, as both groups highlighted different requirements for the tools.\n\nFrom a methodological perspective, the formative design of the study was quite appropriate because the tools take very different approaches and the participants had relatively little prior experience in the subject matter. However, it would have been useful to give all participants a brief introduction to Python and Jupyter notebooks beforehand, so that handling the notebooks would not have interfered with the actual execution of the workflow in OpenVibSpec. Additionally, instructions on how to perform different k-means clusterings in Orange would have been helpful, since none of the participant had tried this on their own. On the other hand, multiple clusterings would have further increased the processing time in Orange.\n\nThe study benefited from the fact that not only one tool was tested by participants, but that participants used two different tools (GUI vs. script-based) and thus also compared advantages and disadvantages of both approaches to implement workflows. Furthermore, clear preferences between user groups could be identified and partial needs could be revealed. For example, the need to train potential users with programming knowledge became clear, although the task in the study could also be solved without programming knowledge.\n\nAnother aspect that could not be considered in this study are learning effects and memorability. Thus, it would be interesting to see how the participants would handle the applications after several weeks, how well they would solve the tasks, and whether the same usability problems would occur again. In addition, one could examine how much learning effort participants without programming expertise would have to put in before they would be able to independently recreate workflows with OpenVibSpec and Orange, and how the perception towards the two tools would change.\n\n\nConclusions\n\nThis study presented findings from a formative usability study that involved two different user groups solving a specific biomedical image analysis task based on two different implementations, one GUI based and one script-based. With the exploratory nature of this study, specific points of improvements for the different workflow implementations regarding different user groups could be identified. One example for such an improvement is the explanation of workflow results. Whereas research directions such as Explainable AI32 focus on making the internal decisions of AI-powered systems more transparent, this study shows that in the field of bioimaging, explaining the output of the systems could be improved by providing useful information to support the interpretation of the results.\n\nA major limitation of this study certainly lies within the limited prior knowledge of the participants. For future studies, more insight will be gained by involving user groups with more prior knowledge. Also, the study setup only allowed basic comparison between the different user groups and the two implementations, again mainly due to the limited prior knowledge of the participants. Extended future studies may overcome this limitation by conducting a community-wide data challenge accompanied by a usability study of selected participating groups. Besides providing access to significant user groups and allowing a more systematic comparison between user groups and tools, such study may also elucidate the inductive bias of different users and tools for workflows which require a human-in-the-loop.\n\n\nData availability\n\nZenodo: A formative usability study of workflow management systems in label-free digital pathology - Data and Code24\n\nThis repository contains the following underlying data:\n\n• Tutorial-Video.mp4 a tutorial video presented to participants at the beginning of the study\n\n• code/openvibspec/code for script based workflow, including a github snapshot of the OpenVibSpec repository\n\n• orange/GUI based workflow\n\n– workflow-instructions-orange.pdf instructions for orange given to participants\n\n– CompleteWorkflowOrange.ows Orange file that contains the full workflow\n\n• data/openvibspec training and test data for use in openvibspec\n\n• data/orange training and test data for use in Orange\n\nZenodo: A formative usability study of workflow management systems in label-free digital pathology - Questionaires30\n\nThis repository contains the following underlying data:\n\n• data.xlsx demographic data of participants, time needed for task, additional notes\n\n• demographics_questionnaire_de.docx demographic questionnaire in German\n\n• demographics_questionnaire_en.docx demographic questionnaire in English\n\n• interview_de.docx interview questions in German\n\n• interview_en.docx interview questions in English\n\n• maxqda_export_de.xlsx MaxQDA Export file with codes and interview statements in German\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\nAM and TH conceived the project. MJ and EF conducted the usability study. APR and JB prepared software and data for the study. MJ, APR, TH and AM analyzed the outcome of the study. MJ, APR, TH and AM wrote and revised the manuscript.", "appendix": "References\n\nDemšar J, Curk T, Erjavec A, et al.: Orange: data mining toolbox in python. J. Mach. Learn. Res. 2013; 14(1): 2349–2353.\n\nBerthold MR, Cebron N, Dill F, et al.: Knime-the konstanz information miner: version 2.0 and beyond. AcM SIGKDD Explorations Newsletter. 2009; 11(1): 26–31. Publisher Full Text\n\nCarpenter AE, Jones TR, Lamprecht MR, et al.: Cellprofiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006; 7(10): 1–11.\n\nGiardine B, Riemer C, Hardison RC, et al.: Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 2005; 15(10): 1451–1455. PubMed Abstract \| Publisher Full Text\n\nKöster J, Rahmann S: Snakemake—a scalable bioinformatics workflow engine. 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PubMed Abstract \| Publisher Full Text\n\nRaulf AP, Butke J, Küpper C, et al.: Deep representation learning for domain adaptable classification of infrared spectral imaging data. Bioinformatics. 2020; 36(1): 287–294. PubMed Abstract \| Publisher Full Text\n\nJelonek M, Raulf A, Fiala E, et al.: A formative usability study of workflow management systems in label-free digital pathology - Questionnaires.February 2022. Publisher Full Text\n\nRandles BM, Pasquetto IV, Golshan MS, et al.: Using the jupyter notebook as a tool for open science: An empirical study. 2017 ACM/IEEE Joint Conference on Digital Libraries (JCDL). 2017; pages 1–2. IEEE.\n\nAdadi A, Berrada M: Peeking inside the black-box: a survey on explainable artificial intelligence (XAI). IEEE access. 2018; 6: 52138–52160. Publisher Full Text" }	[ { "id": "123780", "date": "07 Mar 2022", "name": "Johannes Köster", "expertise": [ "Reviewer Expertise Reproducible data analysis", "bioinformatics. I would like to express though that I am not an expert on user studies at all. Hence", "it should be important to also have a reviewer from that domain!" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe an exemplary usability study on the analysis of label free pathology imaging data. For this purpose, non-experts from two groups with different backgrounds (computer science students and life science students) have been recruited. The participants were asked to complete a data analysis task with (a) a GUI based WMS (Orange), and (b) a scripting language, domain specific WMS (OpenVibSpec). In both cases, participants received instructions on how to build the analysis. The analysis task itself was basically the same, despite some necessary simplifications for the Orange case.\nGeneral comments: The manuscript is well written and understandable. I would like to express that I have expertise in reproducible data analysis, but NOT in usability studies with humans. Hence, this review should be complemented by an expert in this field.\nSpecific comments:\nThe definition of the two terms formative and summative does not become entirely clear. In particular in the case of summative. I suggest outlining both the “goal” and the “method” of each study type separately.\n\nOn Page 7, paragraph 1: What is meant by “at least one participant [...] was able to solve the task”? Why is it impossible to provide the exact number?\n\nI have some problems with the “efficiency” judgement that is extracted from the study. The authors of course do not claim that this is a generalizable outcome of their study, however, I think it should be discussed how exactly efficiency should be defined and how it can best be measured in this context. It seems like the different participants have also different definitions of efficiency in their mind. What measures have to be taken to “normalize” the interpretation of efficiency across participants, either beforehand or retrospectively? Or are there non subjective criteria that could be checked in the study?\n\nI think it would be nice to show more of the study results in a visual way in the paper. What questions were asked and what answers were given (e.g. barplots)? Maybe some visualization showing how the final solutions of the participants differed?\n\nI wonder what number of participants would be needed to draw generalizable conclusions (at least for this specific task/WMS combination). This should be discussed and analyzed. Were the recruited groups large enough? Is it possible to estimate the required size for the future? If not what would be needed to come up with proper estimations?\n\nIt seems that it would also be a good idea to discuss and look for (I know the sample size is very small) batch effects in the study groups. Things like the time of the day (how tired were the participants), the weather (maybe some people did this on a sunny warm day, while others performed the task while it was anyway raining). Can it be excluded that any batch effect was confounded with certain judgements of the participants?\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? Partly", "responses": [] }, { "id": "142157", "date": "05 Jul 2022", "name": "Nelson Pacheco Rocha", "expertise": [ "Reviewer Expertise Usability assessment." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written, and the research question deserves attention. However, the manuscript presents several flaws.\nSection Usability Testing.\nThis section needs to be better structured as it is a bit confusing in terms of some concepts.\nStarting with the title. Usability testing is about usability assessment involving experiences with end-users. However, in the section Usability Testing the authors introduce not only test methods but also inspection methods. Probably, a better name for the section would be usability evaluation or usability assessment.\nThe authors stated, \"in its test design (task-based tests vs. cognitive walkthrough\". This is incorrect since usability evaluations can be performed using test methods or inspection methods. Cognitive walkthrough is just one of the inspection methods. Another one is the heuristic evaluation. In this respect, the sentence \"Besides test-based evaluations, heuristic usability inspections are also commonly used\" should be rewritten.\nStill, in the section Usability Testing, the authors stated, “For example an expert-based usability evaluation can possibly be applied with a smaller number of participants than for example user-based usability tests”. The concept of expert-based usability evaluation was not introduced before and can be misinterpreted. For instance, a usability test of a medical device involving physicians or nurses (which are experts in their domains and, therefore, might be applied the designation expert-based usability evaluation) also requires a significant number of participants. Probably, what the authors want to say is that inspection methods (conducted by usability experts) require a smaller number of participants than usability tests.\nSection Methods\nStudy participants and setup\nIn this subsection, authors should systematize the rationale for the experiment (i.e., two distinct groups of participants) and how they planned to recruit participants.\nOn the other hand, the characteristics of the individuals who participated in the experiments (i.e., the first and third paragraphs of this subsection) are results and as such should appear in the Results section.\nOn the other hand, the methods section lacks important information. The authors state that a “semi-structured interview consisted of general questions about the participants and their prior knowledge, as well as questions integrating Nielsen’s usability heuristics”. However, the Nielsen’s heuristics (i.e., Visibility of system status, Match between system and real world, User control and freedom, consistency and standards, Error prevention, Recognition rather than recall, Flexibility and efficiency of use, Aesthetic and minimalist design, Help users recognize, diagnose, and recover from errors, and Help and documentation) do not match with the dimensions that appear in the results (i.e., Task completion, Task complexity, Comprehension of results, Severe usability issues, Learnability, Efficiency, Error messages, and error prevention). Neither do the “general questions about the participants and their prior knowledge.\nTherefore, in the section methods, the authors should introduce and justify the dimensions that they considered in the Results (i.e., Task completion, Task complexity, Comprehension of results, Severe usability issues, Learnability, Efficiency, Error messages, and error prevention). Moreover, it is required an explanation of how these dimensions were measured.\nSection Results and Discussion\nIn the abstract, the authors stated: “The main intention is to gain first in-sights into the systematic study of usability in the context of biomedical image analysis, and formulate experiences and guidelines for future usability studies dealing with workflow management systems”. Therefore, the study reported in the manuscript has the broad objective to contribute to guidelines for future usability studies dealing with workflow management systems. This is also referred to in the Introduction Section: “may help to summarize experiences to formulate guidelines for future developments”. However, analyzing the remaining sections of the manuscript, this goal is never referred to. Therefore, the authors should either reformulate the objectives in the Abstract and Introduction or introduce in the Results and Discussion sections an analysis of how the study contributes to this broad objective.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-192
https://f1000research.com/articles/11-190/v1	15 Feb 22	{ "type": "Software Tool Article", "title": "SASCRiP: A Python workflow for preprocessing UMI count-based scRNA-seq data", "authors": [ "Darisia Moonsamy", "Nikki Gentle", "Darisia Moonsamy" ], "abstract": "In order to reduce the impact of technical variation inherent in single-cell RNA sequencing (scRNA-seq) technologies on biological interpretation of experiments, rigorous preprocessing and quality control is required to transform raw sequencing reads into high-quality, gene and transcript counts. While hundreds of tools have been developed for this purpose, the vast majority of the most widely used tools are built for the R software environment. With an increasing number of new tools now being developed using Python, it is necessary to develop integrative workflows that leverage tools from both platforms. We have therefore developed, SASCRiP (Sequencing Analysis of Single-Cell RNA in Python), a modular single-cell preprocessing workflow that integrates functionality from existing, widely used R and Python packages, and additional custom features and visualizations, to enable preprocessing of scRNA-seq data derived from technologies that use unique molecular identifier (UMI) sequences in a single Python analysis workflow. We describe the utility of SASCRiP using datasets derived from peripheral blood mononuclear cells sequenced using droplet-based, 3′-end sequencing technology. We highlight SASCRiP’s diagnostic visualizations and fully customizable functions, and demonstrate how SASCRiP provides a highly flexible, integrative Python workflow for preparing unprocessed UMI count-based scRNA-seq data for subsequent downstream analyses. SASCRiP is freely available through PyPi or from the GitHub page.", "keywords": [ "single-cell RNA-seq", "gene expression", "workflow", "Python", "R" ], "content": "Introduction\n\nSince a method for single-cell RNA sequencing (scRNA-seq) was first proposed (Tang et al., 2009), a number of diverse technologies have emerged for studying gene expression at single-cell resolution. Among these, droplet-based, 3′-end sequencing technologies such as Drop-seq (Macosko et al., 2015), inDrop (Klein et al., 2015), and in particular, 10X Genomics Chromium (Zheng et al., 2017) have become increasingly popular for studying gene expression across different cell types and cellular states due to their lower sequencing cost per cell, and higher throughput relative to other available technologies. These technologies rely on the use of short unique molecular identifier (UMI) sequences that are used to both quantify gene expression and reduce technical variation inherently present in scRNA-seq datasets (Islam et al., 2014; Kivioja et al., 2011). However, datasets derived using these UMI count-based technologies need to undergo several preprocessing and quality control steps before they can be used to address biological hypotheses.\n\nAs a result, hundreds of software tools have been developed to perform these preprocessing and quality control steps, with the vast majority of these software tools having historically been built for the R software environment (Zappia et al., 2018). One of the most popular R packages is undoubtedly Seurat (Stuart et al., 2019), which has become an integral component of many scRNA-seq workflows due to the wide range of analysis methods it offers and the utility of its custom data structures. However recently, an increasing number of new tools are being developed using the Python programming language (Zappia & Theis, 2021). One such tool is kb-python (Bray et al., 2016; Melsted et al., 2021), a wrapper for the kallisto \| bustools scRNA-seq workflow (Melsted et al., 2021). Therefore there is a growing need to develop tools that enable integration of widely used existing R-based tools like Seurat and newer Python-based tools and analysis workflows like kb-python.\n\nWe therefore introduce SASCRiP (Sequencing Analysis of Single-Cell RNA in Python), a flexible and modular Python package designed to integrate kb-python, selected Seurat features, and additional custom data processing and visualization functions in a way that simplifies and streamlines preprocessing and quality control of UMI count-based scRNA-seq data in preparation for downstream analyses like clustering, data integration, and differential gene expression analysis.\n\n\nMethods\n\nSASCRiP (Moonsamy, 2021a) implements preprocessing of scRNA-seq datasets through four main functions, namely kallisto_bustools_count, seurat_matrix, run_cqc, and sctransform_normalize (Figure 1).\n\nThe SASCRiP workflow begins with the input of FASTQ files, and a Seurat object and mtx matrix containing gene expression values are produced as output. All SASCRiP functions, namely kallisto_bustools_count, seurat_matrix, run_cqc, and sctransform_normalize, produce intermediate files that are used as input into the following function.\n\nThe kallisto_bustools_count function wraps functions from the kb-python package, which itself wraps functions from the kallisto \| bustools workflow. This function takes as input unprocessed paired-end FASTQ files derived from UMI-based scRNA-seq experiments. These are then, by default, pseudoaligned to a user-specified indexed transcriptome. The user also has the option to generate a new index and transcript-to-genes mapping file if these are not available. All aligned sequences are stored within a barcode UMI set (BUS) file that contains the barcode and UMI sequences, as well as the aligned transcripts. The kallisto_bustools_count function uses BUStools (Melsted et al., 2019) to remove polymerase chain reaction (PCR) duplicates and correct the barcode sequences that differ by 1 hamming distance from a barcode whitelist. The kallisto_bustools_count function includes barcode whitelists for 10x Genomics Chromium v1, v2 and v3, as well as for InDrops v3 data. For other UMI-based technologies, kallisto_bustools_count includes parameters that allow for the use of user-supplied barcode whitelists. By default, the BUS file is filtered to remove barcodes with no corresponding transcript information. Gene-level count matrices are then returned in the matrix market (mtx) format, where cells and genes are represented in rows and columns, respectively.\n\n\n\nSASCRiP also includes an additional, optional function, edit_10xv1_fastq, that transforms scRNA-seq data obtained from experiments using 10x Genomics Chromium v1 technology into a format compatible with the kallisto \| bustools workflow. Although these types of files are already supported by the kallisto \| bustools workflow, most FASTQ files stored in public sequencing data repositories/databases are not provided in the required format. In situations where only two FASTQ files (one containing the transcript and UMI sequences, and the other containing the barcode sequences) are provided, the edit_10vx1_fastq function separates the UMI and transcript sequences into two new FastQ files, so that the three files required (containing the transcript, UMI, and barcode sequences) are available as input for the kallisto \| bustools workflow.\n\n\n\nCount matrices obtained using the kallisto \| bustools workflow cannot be directly imported into Seurat. Therefore the SASCRiP function, seurat_matrix, converts the output obtained from BUStools (where genes are represented as rows and cells as columns) into a format compatible with Seurat, where cells are represented as rows and genes as columns. The seurat_matrix function also converts gene identifiers in the transcript-to-gene index file from the kallisto \| bustools workflow from ENSEMBL IDs to their corresponding HGNC gene symbols, as required by Seurat.\n\n\n\nGene-level count matrices obtained from either Seurat or SASCRiP’s seurat_matrix function can then be used as input for SASCRiP’s run_cqc function, which leverages Seurat’s custom data structures to identify low quality cells. The run_cqc function classifies cells as being of low quality based on two per-cell metrics: the total number of genes detected, and the percentage of sequencing reads that map to mitochondrial genes. These metrics are calculated automatically when a Seurat object is created. By default, run_cqc first removes cells with fewer than 200 genes (Ilicic et al., 2016), then cells where more than 10% of sequencing reads map to mitochondrial genes Osorio & Cai (2021). However, all parameters used to distinguish low quality cells from high quality cells can be defined by the user.\n\nThe run_cqc function also calculates the median absolute deviation (MAD) of the total number of genes per cell, allowing for the identification and removal of cell doublets. SASCRiP prioritises the removal of heterotypic doublets, such that, by default, cells with a total gene count higher than six MAD values from the median are classified as outliers and removed. The MAD values and outlier thresholds are calculated using the following equation:\n\n\n\n\n\n\n\n\n\nHere, x represents the total gene count for all cells, xi is the gene count for a given cell, and n is the number of MAD values used to classify a cell as an outlier. The MAD value cut-off threshold can be manually defined by the user. Alternatively, other outlier detection methods, such as standard deviation, are also included within SASCRiP and can be selected as a substitute to MAD.\n\n\n\nMultiple output files are produced by the run_cqc function. Cell quality control metrics and UMI count data are returned as Seurat objects, in rds format, and a log file is created. The cell quality control metrics can also be returned in tsv format, to facilitate data visualization; and UMI counts can be returned as an mtx matrix, for use with alternative analysis tools. In this way, output from SASCRiP can be easily integrated into alternative workflows and analysis pipelines that require different file formats.\n\nSASCRiP performs normalization and variance stabilization through the sctransform_normalize function, which serves as a wrapper for sctransform (Hafemeister & Satija, 2019). In this way, technical variation present in the dataset due to differences in sequencing depth between cells is reduced. All parameters incorporated within the original sctransform::vst function can be modified through the sctransform_normalize function. Corrected UMI counts are log-normalized in order to obtain gene expression values and the 2000 most variable genes in the dataset are identified and returned. Normalized gene expression values or UMI counts may be returned, either as a Seurat object or as an mtx file.\n\n\n\nThe modularity of SASCRiP allows all functions to be implemented independently, but the package also includes an all-in-one function, sascrip_preprocess, that implements the entire workflow from start to finish. In order to ensure that appropriate quality control measures are applied at each stage of the workflow, quality control metrics are provided at multiple checkpoints in the SASCRiP workflow when the sascrip_preprocess function is executed. These metrics can be printed to the screen while running the workflow and/or written to file for later use.\n\nAll SASCRiP functions are designed and implemented in Python 3 (v3.7 or higher), and the package is available on PyPi. As SASCRiP also incorporates a number of R packages (including Seurat), R (v3.6 or higher) is also required. Any additional R packages required can then be installed through SASCRiP, if needed. SASCRiP was developed primarily for use on Unix-based operating systems, however the source code can be adapted for use on Windows platforms (as described in the SASCRiP documentation). SASCRiP can be used to process scRNA-seq datasets consisting of up to 10 000 cells on a standard laptop with 8 Gb RAM. At least 16 Gb of RAM is recommended for larger datasets.\n\n\nUse cases\n\nIn order to demonstrate the utility of the SASCRiP workflow, we reanalyzed three scRNA-seq datasets derived from peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals (Zheng et al., 2017). Cell counts range from 2000 to 10 000 cells per donor. SASCRiP’s sascrip_preprocess function was used to perform all preprocessing steps required to generate a high-quality gene expression dataset that could then be readily input into Seurat for cell clustering and annotation. All SASCRiP functions were implemented using the default parameters.\n\nAs these datasets were obtained using 10X Genomics Chromium v1 technology, SASCRiP’s edit_10xv1_fastq function was first used to generate the three FASTQ files required by the kallisto_bustools_count function. Gene-level count matrices were then obtained using the kallisto_bustools_count function, and cell quality control metrics were calculated using the run_cqc function (Moonsamy, 2021b). Visualization of these metrics allowed low quality cells (Figure 2) and cell doublets (Figure 3) to be easily identified and removed. These metrics, together with the UMI count data, were then input as Seurat objects, into the sctransform_normalize function. This allowed for normalization and variance stabilization of these UMI counts (Figure 4), so that gene expression could be quantified. In addition, the 2000 most highly variable genes, based on standardized variance, were identified (Figure 5). The normalization data was then output as a Seurat object, in preparation for clustering.\n\nCells that fall (A) below the given threshold for gene count (in this case, 200) and (B) above the maximum threshold for mitochondrial percentage (in this case, 10%) are shown in red.\n\nPotential cell doublets (black dots) are identified relative to the median total gene count per cell. Cells with a total gene count more than six MADs from the median are flagged for removal.\n\nThe total number of UMIs relative to the total number of unique genes detected is visualized before (blue) and after (pink) normalization and variance stabilization using regularized negative binomial regression, as implemented in sctransform (Hafemeister & Satija, 2019). Each point represents a single cell.\n\nThe top 2000 most highly variable genes (purple) are identified based on standardized variance. Each dot represents a gene with its standardized variance shown on the y-axis and the average gene expression (calculated using the normalized gene counts) on the x-axis. The 10 genes with the greatest variance across all cells are labelled.\n\nFinally, to demonstrate the quality of the preprocessed data obtained from SASCRiP, Seurat v3.2.0 was used to cluster the two datasets identified as being of high quality (donors B and C) following processing with SASCRiP. The RunPCA function was used to perform dimension reduction (Stuart et al., 2019), and graph-based clustering was performed using the FindNeighbours function (Xu & Su, 2015). In both datasets, the preprocessed data obtained from SASCRiP produced distinct clusters (Figure 6A), corresponding to major PBMC cell types (Figure 6B), suggesting that SASCRiP produces high quality, preprocessed data, suitable for downstream scRNA-seq analyses and applications.\n\nUniform manifold approximation and projection of ~8000 cells derived from a single donor is shown, coloured according to (A) the clusters identified in the dataset, and (B) the expression of known cell type markers in each cluster. CD3D indicates T cell clusters, MS4A1 B cell clusters, CD68 monocyte clusters, and FCER1A dendritic cell clusters.\n\n\nConclusions\n\nSASCRiP is a Python package that provides a simple, streamlined workflow for preprocessing UMI count-based scRNA-seq data through a series of parameterized functions. To ensure flexibility, these functions allow users to either adopt a set of clearly defined default parameters or to modify any or all of these parameters as they see fit. Also, due to its modular design, SASCRiP’s four major functions (kallisto_bustools_count, seurat_matrix, cell_cqc, sctransform_normalize) can be executed either independently to produce custom visualizations and/or intermediary files that can be used as input for other scRNA-seq tools, or all-in-one to produce high-quality, normalized gene expression datasets that are ready to use for downstream analyses. Collectively, these functions seamlessly integrate the functionality of the widely used R packages, Seurat and sctransform, into a custom Python workflow built around kb-python’s implementation of the kallisto \| bustools workflow. In this way, pseudoalignment, quantification of gene expression, removal of low quality cells and cell doublets, and normalization and variance stabilization can be performed within a single scRNA-seq analysis pipeline.\n\n\nData availability\n\nAll data underlying the results are freely available through 10X Genomics:\n\nDonor A:\n\nhttps://cf.10xgenomics.com/samples/cell-exp/1.1.0/frozen_pbmc_donor_a/frozen_pbmc_donor_a_fastqs.tar\n\nDonor B:\n\nhttps://cf.10xgenomics.com/samples/cell-exp/1.1.0/frozen_pbmc_donor_b/frozen_pbmc_donor_b_fastqs.tar\n\nDonor C:\n\nhttps://cf.10xgenomics.com/samples/cell-exp/1.1.0/frozen_pbmc_donor_c/frozen_pbmc_donor_c_fastqs.tar\n\nZenodo: SASCRiP Supporting Data https://doi.org/10.5281/zenodo.5899870 (Moonsamy, 2021b)\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\nSoftware available from: https://pypi.org/project/SASCRiP\n\nSource code available from: https://github.com/Darisia/SASCRiP\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.5554770 (Moonsamy, 2021a)\n\nLicense: GNU GPLv3", "appendix": "References\n\nBray NL, Pimentel H, Melsted P, et al.: Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016; 34(5): 525–527. PubMed Abstract \| Publisher Full Text\n\nHafemeister C, Satija R: Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 2019; 20(1): 296. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIlicic T, Kim JK, Kolodziejczyk AA, et al.: Classification of low quality cells from single-cell RNA-seq data. Genome Biol. 2016; 17: 29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIslam S, Zeisel A, Joost S, et al.: Quantitative single-cell RNA-seq with unique molecular identifiers. Nat Methods. 2014; 11(2): 163–166. PubMed Abstract \| Publisher Full Text\n\nKivioja T, Vähärautio A, Karlsson K, et al.: Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2011; 9(1): 72–4. PubMed Abstract \| Publisher 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\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\nMelsted P, Booeshaghi AS, Liu L, et al.: Modular, efficient and constant-memory single-cell RNA-seq preprocessing. Nat Biotechnol. 2021; 39(7): 813–818. PubMed Abstract \| Publisher Full Text\n\nMelsted P, Ntranos V, Pachter L: The barcode, UMI, set format and BUStools. Bioinformatics. 2019; 35(21): 4472–4473. PubMed Abstract \| Publisher Full Text\n\nMoonsamy D: SASCRiP (0.1.2). Zenodo. 2021a. http://www.doi.org/10.5281/zenodo.5554770\n\nMoonsamy D: SASCRiP Supporting Data (0.1.2) [Data set]. Zenodo. 2021b. http://www.doi.org/10.5281/zenodo.5899870\n\nOsorio D, Cai JJ: Systematic determination of the mitochondrial proportion in human and mice tissues for single-cell RNA-sequencing data quality control. Bioinformatics. 2021; 37(7): 963–967. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStuart T, Butler A, Hoffman P, et al.: Comprehensive Integration of Single-Cell Data. Cell. 2019; 177(7): 1888–1902.e21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTang F, Barbacioru C, Wang Y, et al.: mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods. 2009; 6(5): 377–382. PubMed Abstract \| Publisher Full Text\n\nXu C, Su Z: Identification of cell types from single-cell transcriptomes using a novel clustering method. Bioinformatics. 2015; 31(12): 1974–1980. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZappia L, Phipson B, Oshlack A: Exploring the single-cell RNA-seq analysis landscape with the scRNA-tools database. PLoS Comput Biol. 2018; 14(6): e1006245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZappia L, Theis FJ: Over 1000 tools reveal trends in the single-cell RNA-seq analysis landscape. Genome Biol. 2021; 22(1): 301. 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. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "123750", "date": "22 Feb 2022", "name": "Fabiola Curion", "expertise": [ "Reviewer Expertise Single cell genomics", "bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have presented a workflow/package to deal with common steps of the pre-processing of single-cell RNA-seq data from raw reads until the normalization step, producing data that can be readily used by downstream tools. Whilst we appreciate it is useful to have a Python interface for some of these steps, it is not clear what is the advantage of using a further wrapper around the existing Python implementation of Kallisto\|Bustools. The second half of the package is a wrapper around the commonly used R tool Seurat but it is not clearly demonstrated how useful it is to wrap Seurat instead of using the equivalent functionality in existing Python packages such as Scanpy. We believe there are some useful additions, such as the conversion of FASTQ files from old chemistry versions to new ones that can be used by Bustools and the transposition of the matrix output from Kallisto\|Bustools into the default Seurat structure (gene x cell), but these could be better emphasised in the text.\nSpecific comments\nThe wrapper around kallisto is not well justified. If the selling point of this solution is to deal with v1 10x chemistry that could be better emphasised.\n\nThe technical description of read sequence structure for 10x and similar droplet-based methods is somewhat lacking clarity. Please provide one or two sentences describing the bioinformatics processing needed to deal with read structures for different chemistries.\n\nThe conversion of Bustools output to Seurat is a good idea. However, this is described twice in the manuscript which is confusing.\n\nWe suggest highlighting that there is a further entry point into the workflow (count matrix as opposed to FASTQ files)\n\nYou suggest a metric for detecting doublets based on outliers in the number of expressed genes but there is no analysis to show how this effectively discriminates known doublets from singlets. We would suggest applying it to one of the datasets used to demonstrate other doublet-detection methods.\n\nIt is not clear if the workflow includes plots as default outputs at any given checkpoint. If this is the case please describe that in the text.\n\nWe found Figure 2 to be quite unclear. Are the left and right columns two different datasets? Why are the scales in panel A different but the same scale is used in panel B? Better labeling of the plots and more detail in the caption would help clarify these issues.\n\nWe could not find code for the analysis presented in the paper. Please include this in a publicly available repository.\nSoftware\nWe were able to install the software using the provided instructions\n\nWe were not able to test the software because no example dataset was provided. We suggest writing a short vignette/tutorial showing how the different parts of the package can be used on a small test dataset.\n\nThere is good function documentation in the GitHub README but we suggest moving this to another format (such as a Read The Docs page) which would be more accessible for users.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] }, { "id": "125623", "date": "14 Mar 2022", "name": "Fangming Xie", "expertise": [ "Reviewer Expertise Bioinformatics", "Neuroscience", "Computational analysis of single-cell transcriptomics and epigenomics data" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMoonsamy and Gentle presented Sequencing Analysis of Single-Cell RNA in Python (SASCRiP), a modular analysis pipeline that processes UMI-based scRNA-seq data from raw sequencing reads to transcript counts, normalization, and visualizations. The software integrated existing tools, including kallisto-bustools (kb python as its Python wrapper) and Seurat (an R package), into a single Python workflow. The authors showcased the usage of their pipeline using public PBMC scRNA-seq datasets.\nStrengths\nOverall, the manuscript was well written and has made its main message clear. The authors did a good job presenting their workflow, functionalities, and results. As a technical manuscript, I liked the code vignettes displayed throughout the main text. Its Github repo also contains a detailed README document.\n\nMajor limitations\nThe software is mainly a Python wrapper around two existing tools: kb-python (in Python) and Seurat (in R). As kb-python is already a Python wrapper of Kallisto, it is not clear to me what this new wrapper has to offer more than the existing one.\n\nThe authors should comment on native Python scRNA-seq analysis packages, such as the widely used Scanpy, and highlight the similarities and differences of their package.\n\nWhile the README file is useful, it would be more helpful if the authors can also include a tutorial or an example script that runs through the workflow with a small chunk of data, as Seurat did.\nMinor comments\nIn Page 5, the authors used MAD to flag potential doublets. The equations for MAD need a bit more explanation/justification. For example, why adding 1.483? What is the unit of x? Is there a citation for this method?\n\nIn Figure 2, it would be helpful to include the number or the fraction of cells in each color/category, in addition to the scatter plots. (In Figure 2A (left), it looks like most cells were low-quality, at least that’s the first impression.)\n\nIn Figure 3, donor A looks very different from the other donors. What was the reason?\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-190
https://f1000research.com/articles/11-189/v1	15 Feb 22	{ "type": "Research Article", "title": "A close-up of the use of weblogs and blogging buddies: A case study in Malaysia", "authors": [ "Yong Eng Chua", "Sareen Kaur Bhar", "Sareen Kaur Bhar" ], "abstract": "Background – Peer review or evaluation has been seen as a promising approach in helping students to learn from one another instead of relying solely on their overwhelmed teachers for feedback on their written work. However, there have been mixed reviews on the effectiveness of this method. This study aims to examine the effects of peer review on blogging buddies’ writing performance as well as to suggest the groundwork needed for an effective way to pair the blogging buddies to ensure that the peers can bilaterally benefit each other.\n\nMethods – A qualitative case study was conducted on eight students from a private university in Malaysia. The writing scores for three different essays (pre and post peer review) were tracked to observe its effects on the respective blogging buddies’ writing performance.\n\nResults – The findings have shown that the students’ proficiency levels played an important part in pairing the students as it impacted their writing performance and the effectiveness of the peer review activity. Poor proficiency students who were paired with average proficiency students seemed to make the greatest progress and were the most prominent beneficiaries. Even though these average students benefited from being a reviewer; this pairing was a disadvantage when they reposted their essays after peer review. For high proficiency students who were paired together, they recorded either an increased score or a same score in their post-peer writing.\n\nConclusions – This study has shown that for peer review via weblogs to be effectively implemented in writing classes, proper groundwork needs to be laid for instructors to ensure that adequate training is provided, the peers are properly paired, and sufficient support is provided to these blogging buddies.", "keywords": [ "Peer Review", "Weblogs", "Writing Skills" ], "content": "Introduction\n\nThe involvement of peers in language learning is indispensable as language learning has become an act of interacting and collaborating among learners as they work and negotiate to form meaning or make sense of what is being taught (Gupta et al., 2019; Richards, 2006). At present, learning activities in class are not drills but pair or group work, role playing or project-based assignments. Learners are supposed to produce work in the second language and make all the mistakes they “need to make” to learn the rules of the language “inductively” (Richards, 2006). The errors made are perceived from a different point of view, and the learners are expected to learn the language rules by getting feedback for their trials and errors. The feedback received is also no longer from their language teachers as they shoulder more responsibilities for their own learning and rely on their peers for support (Gupta et al., 2019; Blackstone et al., 2007). Peer feedback, which is also referred to as peer review, peer editing, peer evaluation, peer response, and peer assessment (Gedera, 2012; Zhang et al., 2014) has helped to effectively supplement teachers' feedback and self-feedback, which is deemed as one of the most important forms of feedback in the context of English as a Second Language (ESL) (Gupta et al., 2019).\n\nBlogging has the greatest pedagogical potential for academic purposes compared to other Web 2.0 tools (Blackstone et al., 2007) and can be a technology tool to be exploited for peer involvement, such as for Written Corrective Feedback (WCF) (Lee, 2020). In Malaysia, Abdullah (2011) indicated that only few empirical studies have investigated the effects of Web-based Asynchronous Peer Feedback (WAPF) on enhancing writing proficiency. Hence, by using weblogs, this study aims to look at the effects of using peer review in the writing performance of second language learners of English in a Malaysian university.\n\n\nMethods\n\nThis study qualifies as a qualitative case study as it was a direct and in-depth analysis of a small sample of participants. The data were also analysed inductively to form a bigger picture from the information collected (Fraenkel et al., 2012; Ary et al., 2006). Moreover, continuous data over a period were collected to ascertain if improvement could be observed in the participants’ writing performance. A purposive, non-random sampling method was used to access a specific subset of participants that fits a particular profile, i.e., Generation Y’s second language (L2) of English who were geographically convenient to be approached.\n\nAn advertisement was posted on the researcher’s social media page inviting volunteers to join a special English writing class as part of a research project. A total of 36 individuals showed interest but due to the inability to find a common time for all, many had to withdraw. Nevertheless, in total, eight students (six Malaysians and two Namibian students) agreed to volunteer for the project that lasted for 28 weeks. Ethical approval (EA0772021) for this study was obtained from Multimedia University, and all 8 participants who volunteered gave their written consent to be involved in the research. The participants were trained for 2 hours a week for a total of 5 weeks. In those preparatory weeks, they were briefed on the structures and requirements of an essay. The students were provided a scoring guide which was an adapted version of the Northwest Regional Educational Laboratory and Reid’s guide (Education Northwest, 2014; Reid, 1993) (see underlying data) (Chua, 2021). Using the scoring criterion, the students were trained via hands-on practices to assess sample essays on their content, organisation, vocabulary, language, and mechanics. The students gave scores to the essays and provided some feedback at the end of the essay. Then the researcher of this study discussed and moderated the marks given so that the students could provide fair, consistent, and accurate feedback and scores. Lee (2020) encouraged teachers to ensure that “opportunities are provided for students to interact with peers and the teacher so that they can engage with Written Corrective Feedback (WCF) at a “deeper level” (p. 5).\n\nAfter the completion of the training, the peers were randomly paired. They proceeded to create their personal blogging accounts on www.blogger.com and wrote their introductory posts which were evaluated by the researcher to gauge their proficiency levels. As a result, four pairs of blogging buddies were created: Pair 1 (Weak+ Average), Pair 2 (Good+ Good), Pair 3 (Average + Average), and Pair 4 (Weak + Average) for the entire duration of the study. It should be noted that there were two pairs of Weak and Average peers.\n\nThe students were asked to post their first essay on their personal blogs, and their respective blogging buddies left feedback and a score based on the scoring guide used during training. The student would then revise and repost the final version of that essay on their blogs. The revised version would reflect the adoption and/or rejection of their peers’ reviews. The researcher gave the bloggers and their buddies a week to complete the entire cycle. There was a total of three cycles as three essays were assigned.\n\nThe researcher with more than 9 years of teaching experience privately assessed and graded the essays twice (The original essay pre peer review and the revised essay post peer review). The same scoring guide was used by the researcher, who did not leave any comments or feedback on the students’ weblogs so that it would not overshadow any feedback provided by their peers.\n\n\nResults and discussion\n\nTable 1 shows the overall scores given to the students’ posts. The English levels indicated the students’ proficiency levels which were determined based on the self-introductory essay posted as their first entry on their personal blogs. The researcher graded the essays with the following grading system: A+ (90-100), A (80 <90), A- (75<80), B+ (70<75), B (65<70), B- (60<65), C+ (55<60), C (50<55), C- (47<50), D+ (44<47) and F (0<44). Students with a score of above 75 were noted as having “Good language proficiency” while students who scored 60 to 74 were categorised as “Average” and those below 60 were considered as “Weak” (see underlying data) (Chua, 2021).\n\nAfter ascertaining their proficiency levels, marks were given for the essays before and after peer review. After the peer review process was carried out for three rounds, it was apparent that peer review worked differently for different pairs. Blackstone et al. (2007) explained that blogging buddies could have a dual role, “as a good conscience”, where the buddy serves a motivational purpose, and a “proof-reader” buddy who acts as a surrogate teacher that teaches and edits the peer’s work (Blackstone et al., 2007).\n\nWeaker students who were paired with buddies with higher proficiency, benefitted from the peers who played the role of proof-readers, as shown by the scores of Pair 1(Table 1). The student with weak proficiency had an increase of 45% to 55% and 58.3% to 66.7% after her Average peer reviewed her first and second essay, respectively. The same can be observed for the weak student in Pair 4. The scores of all three reposted essays after her average peer reviewed her work were increased from 43.3% to 65%, 60% to 61.7% and 65% to 66.7%, respectively. Zhang et al. (2014) mentioned that peer feedback helps students to notice the cognitive gap that exists especially if the L2 learners are unable to identify their own mistakes. Once their weaknesses are identified, these students can improve their work. Lundstrom and Baker (2009) commented that peer review can be very effective, especially for students with lower proficiency. Ge (2011) also highlighted that “the most low-ability students had made good use of peer feedback”.\n\nHowever, the improvement in writing scores of average students depended on the proficiency of their buddies. In Pair 1, the average student did not show any score difference between his original and revised posts after his work was reviewed by his peer with weak proficiency. The average student maintained the score of 68.3% for the second, and 70% for the third essay. The same could be observed in Pair 4 since the average student who was paired with a weak proficiency blogging buddy maintained the same scores for all three essays. This pattern could perhaps be explained by Nassaji & Swain (as cited in Lundstrom & Baker, 2009), who highlighted that if the writer’s Zone of Proximity Development (ZPD) is different from the reviewer, feedback that scaffolds learning may not fully benefit the writer. Hence, these average students who had been paired with a weaker peer might not see their peers’ feedback as significant enough to adjust or improve their subsequent posts.\n\nIn Pair 3 with both peers considered as students with average proficiency, Student 6 decided not to write essays 1 and 3, yet he provided feedback on the first and second essay of Student 5, which resulted in improved scores for those essays. The last essay received no feedback, but Student 5 took the initiative to approach another peer with an average proficiency to review her essay, and she managed to show an improved score (60% to 71.7%) in her final post. Student 5 just completed one essay which showed an increase from 70% to 71.7% after peer review, but he did not post the rest of the essays despite many reminders.\n\nFor students with good proficiency who were paired together, it can be said that these peers would play the role of a good conscience. This form of good conscience motivates good students to be more accountable and to impress or try to be on par with their peers. Hence in Pair 2, an increase can be seen in the scores for Student 3’s first essay (66.7% to 83.3%), and in Student 4’s first and third essay which increased from 80% to 88.3% and 71.7% to 86.7%, respectively. However, it could also be observed that these students may also maintain the same scores for pre and post peer review. For instance, for Student 3, the same scores were recorded pre and post peer editing for the second and last essay. Student 4’s second essay also recorded the same scores. It could be because their first drafts were of good quality, and their peers could have provided more commendations than recommendations. Ge (2011) also found a similar trend in his advanced students whose writings were almost always favourably commented by others, and hence, saw no need to rewrite or make further improvements to their subsequent posts. Hence, the role of their peers was to support and monitor them to ensure that they were performing at the level that they were expected.\n\nThese findings show that blogging and peer review are exciting and useful collaborative writing activities that can help students to enhance their writing skills (Yu & Hu, 2017). In this case study, good proficiency students who were paired with similar peers made sure that their work was revised to get a better score or at least their standard of writing was maintained although their peers might have not made much contribution in their subsequent posts (Ge, 2011). Hence it is suggested that high proficiency students ought to be paired with someone with similar proficiency (Yu & Hu, 2017).\n\nAverage students who were paired with students of similar level were able to improve as shown in Student 5’s performance. Nevertheless, average students who were paired with a weak student might have been helped less by the peer review process regarding scores. Yet, average students who are paired with lower proficiency students should not feel that they have been taken advantage of as the most beneficial aspect of peer review is providing feedback or being a reviewer, instead of receiving feedback, as they will be able to scrutinise their own work better (Yu & Hu, 2017; Lundstrom & Baker, 2009).\n\nStudents with weak proficiency showed encouraging score improvements after being assisted by another peer, especially if their peers had high English proficiency (Allen & Mills, 2016). Ge (2011) also mentioned that the lower proficiency students were the ones who “made the greatest progress” and were “the most prominent beneficiaries”. However, it is suggested that perhaps low proficiency students should not be paired together or with high proficiency students as the help they render each other may be not be bilateral, compared to pairing them with an average proficiency student (Yu & Hu, 2017).\n\n\nConclusions\n\nIt can be concluded that students, regardless of their English proficiency levels, can be positively impacted by having blogging buddies who also serve as their peer reviewers. However, the writing instructor has to ensure that proper groundwork is laid before autonomy is given to the students to carry out this writing activity. Adequate training needs to be provided with sample essays examined and scoring guides used to help students evaluate the different elements of an essay. During the training, the instructor should also carry out open discussions and go through the moderation process with the students while providing constructive feedback and support to the students.\n\nIn addition, proper pairing of peers must be done to ensure bilateral benefit for the blogging pairs. A pre-test that ascertains the students’ proficiency level ensures that the pair’s Zone of Proximity Development is not too different and feedback that scaffolds learning can be given to each other. Students who have similar proficiency levels should be paired together, but students who have lower proficiency levels ought to be paired with students who have an average proficiency level. The instructor would want to avoid pairing students with low proficiency levels together.\n\nWith sufficient training as well as proper pairing, the students will be able to benefit from this writing activity. In addition, weblog’s public nature will further augment the benefits of this writing activity as the students are aware that they are not just writing for their teachers or for their peers, but they are in fact producing a piece of writing that will be presented to everyone in the blogosphere. However, since this was a case study that only involved eight students, the findings of this small sample size cannot be applied to a bigger population of L2 learners. Moreover, since only three essays were given to the students throughout the study, perhaps more rounds of essay writing can be done for a longer period to track and observe further patterns that can be found in their writing performance which is supported by a blogging buddy.\n\n\nData availability\n\nData Archiving and Networked Services (DANZ): A close-up of the use of weblogs and blogging buddies: A case study in Malaysia.\n\nDOI: https://doi.org/10.17026%2Fdans-xvp-kdz2 (Chua, 2021)\n\nThis project contains the following underlying data:\n\nData file 1. (Data Set and Metadata for A Close-up of the Use of Weblogs and Blogging Buddies)\n\nData file 2. (Data Set containing the Writing Scores for Bloggers Before and After Receiving Feedback from Blogging Buddies)\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\nAuthor contributions\n\nChua Yong Eng: Conceptualization, investigation, formal analysis, writing.\n\nSareen Kaur Bhar: Resources, validation, writing (review and editing).", "appendix": "Acknowledgment\n\nThe author would like to thank the eight students from Multimedia University for their contribution towards the smooth completion of the project.\n\n\nReferences\n\nAbdullah R: Types of web-based asynchronous peer feedback (WAPF) in developing writing skills among undergraduate students. International Journal for Educational Studies. 2011; 3(2): 195–204.\n\nAllen D, Mills A: The Impact of Second Language Proficiency in Dyadic Peer Feedback. Lang. Teach. Res. 2016; 20: 498–513. Publisher Full Text\n\nAry D, Jacobs LC, Razavieh A, et al.: Introduction to research in education. Canada: Thomson Wadsworth; 2006.\n\nBlackstone B, Spiri J, Naganuma N: Blogs in English language teaching and learning: Pedagogical uses and student responses. Reflections on English Language Teaching. 2007; 6(2): 1–20.\n\nChua, CYE (Multimedia University): A Close-Up of the Use of Weblogs and Blogging Buddies: A Case Study in Malaysia. DANS. 2021. Publisher Full Text\n\nFraenkel J, Wallen N, Hyun H: How to design and evaluate research in education. 8th ed. New York: McGraw-Hill; 2012.\n\nGe Z-g: Exploring e-learners' perceptions of net-based peer-reviewed English writing. Computer supported collaborative learning. 2011; 6: 75–91. Publisher Full Text\n\nGedera D: The Dynamics of blog peer feedback in ESL classroom. Teaching English with Technology. 2012; 12(4): 16–30.\n\nGupta S, Abdullah F, Li G, et al.: Peer Assessment in Writing: A Critical Review of Previous Studies. Journal of Advances in Linguistics. 2019; 10: 1478–1487. Publisher Full Text\n\nLee I: Utility of focused/comprehensive written corrective feedback research for authentic L2 writing classrooms.J. Second. Lang. Writ. 2020; 49: 100734. Publisher Full Text\n\nLundstrom K, Baker W: To give is better than to receive: The benefits of peer review to the reviewer's own writing.J. Second. Lang. Writ. 2009; 18: 30–43. Publisher Full Text\n\nEducation Northwest: 2014. 6+1 Trait Rubrics. Reference Source\n\nReid J: Teaching ESL Writing.Longman Publishing Group; 1993.\n\nRichards J: Communicative Language Teaching Today. New York: Cambridge University Press; 2006.\n\nYu S, Hu G: Can higher-proficiency L2 learners benefit from working with lower-proficiency partners in peer feedback?. Teach. High. Educ. 2017; 22(2): 178–192. Publisher Full Text\n\nZhang H, Song W, Shen S, et al.: The effects of blog-mediated peer feedback on learners' motivation, collaboration and course satisfaction in a second language writing course. Australas. J. Educ. Technol. 2014; 30(6): 670–685. Publisher Full Text" }	[ { "id": "123739", "date": "28 Feb 2022", "name": "Pramela Krish", "expertise": [ "Reviewer Expertise Online Language learning" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract\n\nSome implications of the findings need to to be given in the conclusion.\n\nKeywords\n\nAdd one or two more keywords to reflect the paper better.\n\nThe Introduction\nProvides the background of the study. However, the authors have not reviewed the relevant literature for this paper. Literature on Peer Review, Online Peer Review, and Feedback provide a better strength to the paper.\n\nThe ZPD could be the theoretical underpinning for this study and could be explained in relation to this study earlier in the paper.\n\nWhat is the research gap? Why was this student warranted? This is not explained.\n\nMethodology\nWell explained but the last paragraph ---- the researchers?\n\nWhy do you privately assessed and graded the essays? The reason for doing this must be stated clearly.\n\nThe conclusion\n\nCould carry the implications of the study, suggestions and recommendations. This will enable readers to practise this.\n\nThe references must be updated. There are numerous works from 2014 -2020 on weblogs and feedback. Kindly refer to the relevant studies. Check references both the list and in-text citations.\nPaper is well-written but reorganizing some information as discussed in the review may help.\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? Partly", "responses": [] }, { "id": "174993", "date": "05 Jun 2023", "name": "Atik Umamah", "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\nAbstract The abstract is complete; however, it is better to provide more detailed information in the Method Part, such as the instruments and data analysis.\nKeywords It is better to add ‘feedback’ in the keywords since it is the theoretical framework.\nIntroduction The Introduction Part is too short and needs more explanation regarding the theoretical framework. The authors can add an explanation about writing as process which involves revising and editing processes in which feedback is provided. It is also necessary to give more explanations related to the theory of scaffolding and feedback. A review of the previous studies needs to be explained more thoroughly using current publications to identify the research gap.\nMethodology The Methodology Part is well explained; however, it is better to involve an inter-rater when assessing the students’ writing to achieve reliability. If it is impossible, the authors need to provide justifications for not doing so.\nResults and Discussions The results and discussions are complete; however, the authors should explain the results more precisely regarding the writing aspects in that the students give feedback. Additionally, using more current publications within the last five years to compare the current and previous research is also recommended.\nConclusions\nThe conclusions and recommendations are well presented.\nReferences The authors should add more current references published within the last five years to ensure novelty.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "176572", "date": "19 Jun 2023", "name": "Maria B. Cequeña", "expertise": [ "Reviewer Expertise ESL", "Reading comprehension", "flipped learning", "metacognition and comprehension" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 – Add pedagogical implications in the abstract.\n\nIntroduction is not fully developed. Revise it considering the following:\n\nProvide in-depth discussion of Peer feedback in the introduction and relate it to the stages of the writing process.\n\nDiscuss in-depth how blogging can be used as a platform in developing students’ writing through peer feedback.\n\nMake sure to include in your Introduction/Literature Review the in-text citations you used in the Results and Discussion section of your article.\n\nAdd more literature and studies in your Introduction to be able to establish research gap. You may look into the following articles: https://www.researchgate.net/publication/253967244_Peer_feedback_The_learning_element_of_peer_assessment https://educationaltechnologyjournal.springeropen.com/articles/10.1186/s41239-016-0017-y https://www.semanticscholar.org/paper/Using-Peer-Feedback-to-Enhance-the-Quality-of-An-Ertmer-Richardson/3bb8dfd6ce9228976a81fd576533f0d792909692\n\nMake the introduction cohesive by using transitions.\nMethodology\n\nAdd in the procedure how the students were trained in providing feedback.\n\nTo make the study reliable, there should be interrater who needs to rate the essays besides the researcher.\n\nInclude in the methodology how the proficiency level of each participant was measured.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-189
https://f1000research.com/articles/11-188/v1	15 Feb 22	{ "type": "Research Article", "title": "In silico screening of chloroquine analogues for compounds with more affinity for the Plasmodium falciparum chloroquine transporter as potential antimalarial drugs", "authors": [ "Filex Otieno", "Michael Walekhwa", "Filex Otieno" ], "abstract": "Background: Malaria is an acute febrile illness affecting over 229 million people worldwide. Children aged five years and below are affected the most, with the highest prevalence in Sub-Saharan Africa. Chloroquine was previously used as the first-line treatment for malaria due to its affordability and high efficacy, but resistance has developed. Resistance to chloroquine is due to a mutation in the protein Plasmodium falciparum Chloroquine Transporter (pfCRT) which effluxes the drug from the parasitic digestive vacuole, decreasing the drug concentration. Resistance has however been shown to be reversible by compounds that can bind to the protein. Methods: In silico screening for chloroquine analogues was done using SwissSimilarity, SWISSADME, SwissTargetPrediction, Pubchem sketcher, Chimera and Avogadro tools to predict pharmacodynamics and pharmacokinetic profiles of the selected analogues. Results: About 20 compounds with a similarity index of > 95% were obtained from the ZINC database. In total, 12 of the 20 compounds showed a higher binding affinity to the mutant pfCRT protein. Overall, four of the 12 had a binding affinity less than -8.0 compared to -7.0 for chloroquine. Compound ZINC01596768 had the greatest binding strength at -8.3. The other analogues were ZINC38050614, ZINC38050617, and ZINC38050615 with binding interaction strengths of -8.0, -8.2 and -8.2 respectively. Pharmacokinetic profile prediction showed all 12 compounds inhibited the enzymes CYP1A2 and CYP2D6, followed the Lipinski rules, had a high GI absorption, were permeant to the blood brain barrier, had no alerts on the PAINS criteria and had violated the rule of XLOGP3 > 3.5 in lead likeness. Compounds ZINC38050614, ZINC38050617, and ZINC38050615 were predicted to be substrates of P-glycoprotein. The synthetic accessibility score for the twelve compounds were below 3.07. Conclusions: Results demonstrated that the compounds ZINC01596768, ZINC38050614, ZINC38050617, and ZINC38050615 were potential candidates that could be tested and developed as co-formulations of chloroquine.", "keywords": [ "chloroquine", "chloroquine analogue", "SwissSimilarity", "SWISSADME", "SwissTargetPrediction", "Pubchem sketcher", "chimera", "Avogadro", "pharmacokinetic", "pharmacodynamics", "resistance", "pfCRT" ], "content": "Introduction\n\nMalaria is an acute febrile infection caused by Plasmodium parasites. The parasite is transmitted through the bite of an infected female Anopheles mosquito when it is feeding on human blood. Five species of plasmodium parasites exist of which Plasmodium falciparum and Plasmodium vivax have been shown to cause the greatest morbidity and mortality in humans. Malaria cases in the African, South East Asian and Eastern Mediterranean region sare commonly caused by the P. falciparum species while the P. vivax species has been implicated mostly with malarial cases from the American region (Ocan et al., 2019).\n\nMalaria is an infection that is most prevalent in tropical regions across the world. According to the World Health Organization (WHO) report on malaria disease burden as of 2019, there were 229 million cases reported worldwide, which was an increase by one million cases from the previous year (WHO, 2021). Worldwide mortality due to malaria in 2019 was at 409,000, representing a decrease by 2,000 from the year 2018. Children less than 5 years of age across the world accounted for 67% of the total cases and deaths. Moreover, the African region represented the greatest proportion of these cases and deaths, contributing to approximately 94% of the total cases as of 2019 (Fact sheet about malaria, WHO, 2021).\n\nPreviously, chloroquine was the first-line agent for treating all forms of malaria due to its affordability, usability and high efficacy but with time, the parasites P. falciparum and P. vivax developed resistance to it (Al-Bari, 2015). Currently, the first-line agent for malaria involves using artemisinin-based combination therapy (ACT). Though these drug combinations currently in use are effective, they are costly and have a high pill burden. Resistance to chloroquine by P. falciparum was first isolated from South East Asia and it spread across the world. Scholars suggest that resistance to chloroquine led to a doubling of deaths associated with malaria within the sub-Saharan Africa (Kim et al., 2020).\n\nResistance to chloroquine by P. falciparum has been shown to be mediated by the mutant form of the protein P. falciparum chloroquine-resistant transporters (pfCRT) which belongs to the superfamily of transporters called the drug/metabolite transporter (DMT) superfamily (Kim et al., 2020). The protein is a ten transmembrane helix spanning the membrane of the digestive vacuole of the parasite with five helical hairpins. Helices 1-4 and 6-9 form a central cavity acting as a channel for removing molecules from the vacuole (Kim et al., 2020).\n\nThe mechanism of resistance by this mutant protein involves actively binding chloroquine and subsequently effluxing it from the vacuole, leading to a decrease in chloroquine concentration within the vacuole. Chloroquine-associated mutations occur in the K76T region of amino acids which is located directly in the lining of the central cavity (Ocan et al., 2019). These mutations cause an increase in the affinity of chloroquine-binding, leading to increase in efflux rate. Moreover, pfCRT mutations have been shown to also induce resistance to other antimalarial drugs such as the quinolone derivatives (Sanchez et al., 2019). Other studies have shown that drugs such as verapamil reduce the binding of chloroquine to the protein causing a reversal in its resistance nature (Bellanca et al., 2014).\n\nThis study aimed to identify compounds with higher binding affinity to the mutant pfCRT and, in addition, a high or similar antimalarial efficacy as chloroquine, so as to reduce efflux of the parent drug (chloroquine) from the parasitic digestive vacuole while also exacting antimalarial activity. The study will focus on compounds with chloroquine similarity percentage of 95% and above.\n\n\nMethods\n\nIdentification of compounds similar to chloroquine was done through in silico search where a ligand-based virtual screening for chloroquine analogues was carried out on the ZINC database (ZINC15). The search was done by feeding the canonical smiles of chloroquine into SwissSimilarity and running it. The search is based on finding compounds with similar pharmacophore, molecular fingerprint and shape of the parent drug.\n\nCompounds from the search results with a similarity index of 95% were isolated and then drawn using Pubchem sketcher V2.4. Three-dimensional models of the compounds were generated through Avogadro version 1.1.0 after which they were prepared through optimization in Avogadro and minimization in Chimera version 1.14c. The 3D structure of the pfCRT protein was retrieved from the Protein Databank-101 (accession number: 2B9L https://www.rcsb.org/) and standardized using Chimera 1.14c. Finally, surface binding analysis of the prepared compounds was carried out by docking them with the standardized pfCRT protein. The docking scores showing the binding strength of the compounds with the protein were generated and compared with that of chloroquine.\n\nIn addition, prediction of the pharmacokinetic profile of the selected compounds was done using the web tool SWISSADME and also prediction of other likely binding sites of the drug within the body was carried out using the web tool SwissTargetPrediction.\n\n\nResults\n\n\n\n\nDiscussion\n\nP. falciparum resistance to chloroquine is not attributed to drug modification or inactivation, but rather due to increase in efflux of the drug from the digestive vacuole of the parasite (Chinappi et al., 2010; Sidhu et al., 2002). This decreases the concentration of the drug within the vacuole such that it becomes ineffective in completely inhibiting incorporation of heme to hemozoin. The mutant pfCRT protein also binds verapamil and desipramine resulting in reversing of the resistance induced by the mutant pfCRT (Bellanca et al., 2014; Lakshmanan et al., 2005). However, the challenge of using these chemo-sensitizers is that a higher concentration is required to achieve optimal reversal effect and this higher concentration is associated with more adverse effects, seemingly because they are already established drugs acting on different receptors for different functions (Martin et al., 1987).\n\nSince the mutant pfCRT resistance can be reversed by binding to other compounds, we hypothesized that developing a chloroquine analogue with a higher binding affinity for the protein could be co-administered with the parent compound itself (chloroquine) (Kim et al., 2019; Patel and Roy, 2021). This in turn will lead to competitive racing for the binding site and subsequent elimination from the vacuole. The chloroquine analogue with a high affinity and binding capacity for protein will competitively inhibit binding of chloroquine onto the protein, decreasing its efflux from the vacuole. As such, the concentration of chloroquine will be maintained and even elevated, ensuring maximal efficacy. Furthermore, the fact that these analogues are similarly related to chloroquine means they will also prevent incorporation of heme to hemozoin, adding to the efficacy of the parent compound.\n\nTwenty compounds out of the ZINC database search results had a chloroquine similarity index of > 95%. Twelve of these compounds showed a higher binding affinity to the mutant pfCRT protein. Four of these twelve compounds had binding affinity less than -8.0 compared to -7.0 of chloroquine with the compound ZINC01596768 having the greatest binding strength of -8.3. This strong interaction between the protein and the analogues could be useful in ensuring competitive inhibition of pfCRT binding. The other analogues were ZINC38050614, ZINC38050617, and ZINC38050615 with binding interaction strengths of -8.0, -8.2 and -8.2 respectively.\n\nThe pharmacokinetic profile prediction showed all the twelve compounds followed Lipinski rules, had no alerts on the PAINS criteria and in terms of lead likeness, they had violated the rule of XLOGP3 > 3.5 (Daina et al., 2017). They also had a high GI absorption, and thus can be formulated as oral drugs. However, three of these twelve compounds (ZINC38050614, ZINC38050617, and ZINC38050615) are predicted to be substrates of P-glycoprotein and thus, even though they had a high GI absorption, their bioavailability could be reduced due to efflux by the glycoprotein (Kwon et al., 2004). All the compounds were predicted to be permeant to the blood brain barrier and could potentially cause CNS side effects. When it comes to CYP450 inhibition, all the twelve compounds inhibited the enzymes CYP1A2 and CYP26. In contrast to this, all the twelve compounds did not inhibit CYP1A2 but showed mixed results when it comes to inhibition of CYP2C19. Compounds ZINC01873617 and ZINC96331701 did inhibit CYP3A4 while the rest of the compounds did not inhibit the enzyme. The Lipophilicity log P value between 1 and 4 usually provides more optimal physiochemical properties (Benet et al., 2016). Of the twelve selected compounds, compound ZINC01596768, which had the highest docking score, had a log P of 4.15 which, is slightly above the optimal range.\n\nThe synthetic accessibility score for the twelve compounds were all below 3.07, nearing one and as such were easy to synthesize in the laboratory (Daina et al., 2017). Other target prediction by the SwissTargetPrediction tool showed that the compounds interacted less with receptors or enzymes within the body. The compound ZINC01596768 with the highest docking score showed a probability of more than 19% of binding to histamine N-methyl-transferase, histamine 3 receptor and Quinone reductase 2 enzyme. The remaining three compounds with docking score less than -8.0 only interacted with histamine 3 receptor at a probability of more than 19%.\n\n\nConclusions\n\n\n\nI. 12 out of 20 compounds showed greater binding affinity for the mutant pfCRT protein than chloroquine.\n\nII. ZINC01596768 had the greatest binding affinity of -8.3.\n\nIII. ZINC01596768 on simulation had no alerts on PAINS criteria, followed Lipinski rules, had high GI absorption, permeated BBB, inhibited CYP1A2 and CYP2D6 and a log P of 4.15.\n\nThus, compound ZINC01596768 could potentially be co-formulated with chloroquine in the treatment of malaria in chloroquine- resistant cases.\n\n\nData availability\n\nHarvard Dataverse: Insilco Screening of Chloroquine Analogues for Compounds with More Affinity for the Plasmodium falciparum Chloroquine Transporter as Potential Antimalarial Drugs, https://doi.org/10.7910/DVN/OAU1WP (Otieno and Walekhwa, 2022).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAl-Bari MA: Chloroquine analogues in drug discovery: New directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases. J. Antimicrob. Chemother. 2015; 70(6): 1608–1621. PubMed Abstract \| Publisher Full Text\n\nAvogadro: an open-source molecular builder and visualization tool. Version 1.1.0.\n\nBellanca S, Summers RL, Meyrath M, et al.: Multiple drugs compete for transport via the plasmodium falciparum chloroquine resistance transporter at distinct but interdependent sites. J. Biol. Chem. 2014; 289(52): 36336–36351. PubMed Abstract \| Publisher Full Text\n\nBenet LZ, Hosey CM, Ursu O, et al.: BDDCS, the rule of 5 and drugability. Adv. Drug Deliv. Rev. 2016; 101: 89–98. PubMed Abstract \| Publisher Full Text\n\nBerman HM, Henrick K, Nakamura H: Announcing the worldwide Protein Data Bank. Nat. Struct. Biol. 2003; 10(12): 980. PubMed Abstract \| Publisher Full Text Reference Source\n\nChinappi M, Via A, Marcatili P, et al.: On the mechanism of chloroquine resistance in plasmodium falciparum. PLoS One. 2010; 5(11): e14064. PubMed Abstract \| Publisher Full Text\n\nDaina A, Michielin O, Zoete V: SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017; 7(1). PubMed Abstract \| Publisher Full Text\n\nKim J, Tan YZ, Wicht KJ, et al.: Structure and drug resistance of the plasmodium falciparum transporter PfCRT. Nature. 2019; 576(7786): 315–320. PubMed Abstract \| Publisher Full Text\n\nKim J, Zi Tan Y, Wicht KJ, et al.: Structure and drug resistance of the plasmodium Falciparum transporter PfCRT. Biophys. J. 2020; 118(3): 523a. Publisher Full Text\n\nKwon H, Lionberger RA, Yu LX: Impact of P-glycoprotein-Mediated intestinal efflux kinetics on oral bioavailability of P-glycoprotein substrates. Mol. Pharm. 2004; 1(6): 455–465. PubMed Abstract \| Publisher Full Text\n\nLakshmanan V, Bray PG, Verdier-Pinard D, et al.: A critical role for PfCRT K76T in plasmodium falciparum verapamil-reversible chloroquine resistance. EMBO J. 2005; 24(13): 2294–2305. PubMed Abstract \| Publisher Full Text\n\nMartin SK, Oduola AM, Milhous WK: Reversal of chloroquine resistance in plasmodium falciparum by Verapamil. Science. 1987; 235(4791): 899–901. Publisher Full Text\n\nOcan M, Akena D, Nsobya S, et al.: Persistence of chloroquine resistance alleles in malaria endemic countries: A systematic review of burden and risk factors. Malar. J. 2019; 18(1): 76. PubMed Abstract \| Publisher Full Text\n\nOtieno F, Walekhwa M: Insilco Screening of Chloroquine Analogues for Compounds with More Affinity for the Plasmodium falciparum Chloroquine Transporter as Potential Antimalarial Drugs.2022. Harvard Dataverse, V1, UNF:6:gNGxSedlCI4DuM2Uku2m8g== [fileUNF]. Publisher Full Text\n\nPatel C, Roy D: A computational study of molecular mechanism of chloroquine resistance by chloroquine resistance transporter protein of plasmodium falciparum via molecular modeling and molecular simulations. Physchem. 2021; 1(3): 232–242. Publisher Full Text\n\nSanchez CP, Moliner Cubel S, Nyboer B, et al.: Phosphomimetic substitution at ser-33 of the chloroquine resistance transporter PfCRT reconstitutes drug responses in plasmodium falciparum. J. Biol. Chem. 2019; 294(34): 12766–12778. PubMed Abstract \| Publisher Full Text\n\nSidhu AB, Verdier-Pinard D, Fidock DA: Chloroquine resistance in plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science. 2002; 298(5591): 210–213. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Fact sheet about malaria. World Health Organization; 2021, December 6. Reference Source" }	[ { "id": "128920", "date": "29 Jul 2022", "name": "David A. Fidock", "expertise": [ "Reviewer Expertise Plasmodium falciparum", "malaria", "drug resistance", "drug discovery", "genetics", "genomics", "transporters", "mode of action", "therapeutics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 by Otieno and Walekhwa is an interesting study that uses in silico screening to search for compounds that might bind to the Plasmodium falciparum chloroquine resistance transporter PfCRT and therefore prove useful to combine with antimalarial drugs to which PfCRT mediates resistance via a gain of efflux. While the study is purely computational and predictive, the findings are interesting and it is a useful starting point for further studies.\nThe authors should update their introduction to cite most recent WHO data on malaria disease burden (see numbers for the year 2020).\n\nHow many compounds did they examine in the ZINC database?\n\nOne important improvement to the manuscript would be to also show structures in a Figure. This would show the reader if these all share a 4-aminoquinoline ring and how structurally related they are.\n\nThese data are predictive of binding but there is no experimental evidence to support this. The authors should list in their conclusions that the compounds were predicted to have greater binding affinity for the mutant pfCRT protein.\n\nThe authors should also state that further studies are required to test these compounds for activity against Plasmodium falciparum chloroquine-resistant and chloroquine-sensitive parasites cultured in vitro.\n\nThe authors should also indicate that further work is needed to measure the binding affinity of these compounds to purified PfCRT, as was described in the Kim et al. 2019 article that they cite.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "168754", "date": "14 Apr 2023", "name": "Rajalakshmanan Eswaramoorthy", "expertise": [ "Reviewer Expertise Medicinal and Computational Chemistry", "Advanced Biomaterials", "Tissue Engineering and Regenerative Medicine." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented their research work titled \"In silico screening of chloroquine analogues for compounds with more affinity for the Plasmodium falciparum chloroquine transporter as potential antimalarial drugs\" Overall, the article is well written. However, a few issues need to be corrected before it is Approved.\nComments:\nThe article requires thorough grammar and spell check.\n\nThe abstract section needs to highlight the critical finding of the article (numeric values and significance).\n\nThe references section needs to be strengthened in all sections.\n\nThe authors need to provide the selected compounds' 2D structures.\n\nThe results section needs to revise with clarity. The authors provided only tables.\n\nThe rationale behind this present investigation is not clear. The introduction section needs to be revised to emphasize the motivation behind this study.\n\nThe methodology section needs to be revised carefully.\n\nMolecular docking grid box values were not given.\n\nDid the authors do blind docking validation or re-docking to confirm the active site?\n\nIs there any charges added to protein? What is Kollman's charge here?\n\nThe conclusion needs to be revised. Highlight the critical finding and comparative results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-188
https://f1000research.com/articles/10-1128/v1	08 Nov 21	{ "type": "Research Article", "title": "Rice husk and melaleuca biochar additions reduce soil CH4 and N2O emissions and increase soil organic matter and nutrient availability", "authors": [ "Nam Tran Sy", "Thao Huynh Van", "Nguyen Huu Chiem", "Cong Nguyen Van", "Tarao Mitsunori", "Nam Tran Sy", "Nguyen Huu Chiem", "Cong Nguyen Van", "Tarao Mitsunori" ], "abstract": "Background: Biochar is a promising material in mitigating greenhouse gases (GHGs) emissions from paddy fields due to its remarkable structural properties. Rice husk biochar (RhB) and melaleuca biochar (MB) are amendment materials that could be used to potentially reduce emissions in the Vietnamese Mekong Delta (VMD). However, their effects on CH4 and N2O emissions and soil under local water management and conventional rice cultivation have not been thoroughly investigated. Methods: We conducted a field experiment using biochar additions to the topsoil layer (0-20 cm). Five treatments comprising 0 t ha-1 (CT0); 5 t ha-1 (RhB5) and 10 t ha-1 (RhB10), and 5 t ha-1 (MB5) and 10 t ha-1 (MB10) were designed plot-by-plot (20 m2) in triplicates. Results: The results showed that biochar application from 5 to 10 t ha-1 significantly decreased cumulative CH4 (24.2 – 28.0%, RhB; 22.0 – 14.1%, MB) and N2O (25.6 – 41.0%, RhB; 38.4 – 56.4%, MB) fluxes without a reduction in grain yield. Increasing the biochar application rate further did not decrease significantly total CH4 and N2O fluxes but was seen to significantly reduce the global warming potential (GWP) and yield-scale GWP in the RhB treatments. Biochar application improved soil Eh but had no effects on soil pH. Whereas CH4 flux correlated negatively with soil Eh (P < 0.001; r2 = 0.552, RhB; P < 0.001; r2 = 0.502, MB). The soil physicochemical properties of bulk density, porosity, organic matter, and anaerobically mineralized N were significantly improved in biochar-amended treatments, while available P also slightly increased. Conclusions: Biochar supplementation significantly reduced CH4 and N2O fluxes and improved soil mineralization and physiochemical properties toward beneficial for rice plant. The results suggest that the optimal combination of biochar-application rates and effective water-irrigation techniques for soil types in the MD should be further studied in future works.", "keywords": [ "Biochar amendment", "conventional rice farming", "greenhouse gas emissions", "melaleuca biochar", "rice-husk biochar", "soil fertility" ], "content": "Introduction\n\nIn Vietnam, the agricultural sector contributes approximately 30% of national greenhouse gases (GHGs) emissions (MORNE, 2017). For rice cultivation, paddy fields are the primary source of GHGs emissions (Nan et al., 2020; Shinoda et al., 2019), accounting for 50% of the sub-sectors in agricultural production and roughly 14.6% of national GHG emissions in Vietnam (MONRE, 2017). According to NDC (2020), Vietnam is committed to reducing 8% of total national GHGs emissions from domestic resources by 2030. Management and technological strategies will play a vital role in reducing the total carbon footprint. Biochar is a carbonized biomass product produced from thermochemical conversion of organic materials under oxygen-limited conditions (Lohri et al., 2016; Wu et al., 2012; Waqas et al., 2018). Biochar applications have been noted as one of the most promising approaches for reducing GHGs emissions from rice production (Koyama et al., 2015; Wu et al., 2019a; Nan et al., 2021), and IPCC recently recommended the method (Ji et al., 2020). Previous studies have demonstrated that biochar incorporated into soil paddy fields positively rehabilitated soil properties such as pH neutralization, cation exchange capacity (CEC), and buffering capability, soil organic materials (SOM), and nitrogen storage (Qin et al., 2016; Luo et al., 2020); improved plant available water, microporosity, and soil aggregate stability, and decreased bulk density (Burrell et al., 2016); effected on soil functions and fertility (Giagnoni et al., 2019; Siedt et al., 2021); and ameliorated nutrient availability of carbon (C), nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg) and Calcium (Ca) (Li et al., 2019). Furthermore, biochar forms a great habitat for different microorganisms via providing macro-, meso- and micropores (Palansooriya et al., 2019; Wu et al., 2019a), supports microbial communities by providing labile C substrates for degradation (Smith et al., 2010), stimulating biodiversity and abundance of methanotrophic microbes (Qin et al., 2016). Moreover, the addition of biochar to the soil reduces GHGs emissions (Spokas and Reicosky, 2009; Koyama et al., 2015; Nan et al., 2020; Huang et al., 2019) and increases rice yield under different favorable conditions (Yang et al., 2019; Paiman and Effendy, 2020).\n\nIn the Mekong Delta (MD), melaleuca is an abundantly available hard firewood resource, accounting for 176,295 ha (GIZ, 2009); the wood reserve of melaleuca is estimated at 13 million m3. In addition, rice husk is known as a by-product of rice production, accounting for 20% of rice yields (Chungsangunsit et al., 2009). It is estimated that VMD annually produces around 1.9 million tons of rice husk (Son et al., 2017). Biomass (hardwood and crop residues) are often used as typical feedstock for making biochar pyrolysis owing to their multiple-porous structure (Nguyen et al., 2018; Nan et al., 2020), which facilitates the multifunctional purposes of soil amendment and pollutant remediation. Therefore, both melaleuca and rice husk could be used to produce biochar, which is then applied to rice paddy fields as a GHGs emission reduction strategy. Although previous studies have demonstrated the effectiveness of biochar incorporation on reducing GHGs emissions, little attention has been paid to the quantitative variation of rice husk biochar (RhB) and melaleuca biochar (MB) on GHGs emissions and soil improvement in VMD lowland conditions. Moreover, the majority of previous studies exclusively emphasize CH4 and N2O emissions on water practices by controlled irrigation, and alternative wetting and drying, and midseason drainage (Yang et al. 2019, Sriphirom et al. 2020, Uno et al. 2021), while atypical water irrigation regime has not been thoroughly elucidated.\n\nThus, we aimed (i) to elucidate the CH4 and N2O emissions and global warming potential (GWP) from the incorporation of RhB and MB into the paddy field soils under locally typical water management regimes in the VMD, and (ii) to determine the effects of RhB and MB amendments on soil physicochemical properties. We, therefore, conducted a field experiment with a variety of RhB and MB amendment amounts under conventional farming practices. Our field experimentation confirmed that RhB and MB application to rice paddy fields was feasible in reducing GHGs emissions. Simultaneously, biochar application improved soil availability of SOM and anaerobically mineralized N.\n\n\nMethods\n\nA field experiment was carried out on a typical smallholding farmer's paddy field in Thoi An Dong Village, Can Tho city, Vietnam (10°3′44″N, 105°41′55″E). The study area was located in the center of the Mekong Delta, Vietnam, which is a tropical area influenced by the monsoon climate zone, with measured mean annual rainfall (2,088.4 mm), air temperature (27.5 – 27.5 °C), humidity (78.0 – 86.0%), sunshine (2,467.4 – 2,695.4 hours) in the period from 2015-2019 (DONRE, 2020). The precipitation and temperature during the experiment were recorded by a weather station placed at the farmer's house (~150 m from the field experiment). The soil was classified as Thionic Glycesol (International Union of Soil Sciences (IUSS) working group World Reference Base (WRB), 2015) (Dong et al., 2012, Minamikawa et al., 2021). The elementary properties were (0-20 cm depth) as follows: pH (H2O), 5.41; EC, 0.9 mS cm−1; bulk density, 0.92 g.cm−3, silty clay texture (59.3% clay, 39.5% silt, 1.2% sand); organic matter, 87 g kg−1; total N, 4.21 g kg−1; cation exchange capacity (CEC), 37.4 meq 100 g−1; exchangeable K, 0.54 meq 100 g−1, exchangeable Mg, 5.47 meq 100 g−1; exchangeable Ca, 10.5 meq 100 g−1; and total C, 40.76 g kg−1.\n\nRhB was made on-site using a simple semi-industrial pyrolysis batch method (Oikawa et al., 2016). Here a short iron bar was to set onto the ground. A stainless chimney pipe 1.5m long was vertically erected to the bar using wire. The pipe was kept at a 10-cm distance from the ground to release smoke generated during the pyrolysis process. Embers were placed adjacent to the bar to kick off the carbonization process. Then, rice husk was poured around the bar according to a coniform shape with 1.5 m height and 1.5 m diameter. RhB was generated from the bottom to the summit. After finishing the pyrolysis process (two days), RhB was watered to achieve ambient temperature.\n\nMB was produced by a poor-oxygen pyrolysis process under a traditional bell-shaped charcoal production kiln for a 30-day batch. The kiln was made from baked bricks, clay, and sand mortar. The kiln’s structure comprises a bell-shaped heating firewood chamber, a door used for firewood loading, and biochar unloading. A combustion chamber provided hot air for the carbonization process, while four chimneys were installed around the heating chamber discharging smoke during the carbonization process. Firewood was fully loaded according to each layer underneath the heating chamber; the lowest layer was kept 10 cm away from the ground to ensure air convection. Before starting, the door was closed to begin the carbonization process. Air heating from the combustion chamber was slowly provided to the inner heating chamber to form carbonization. After 30 days of pyrolysis, the heating was switched off, and the combustion chamber was blocked off for an additional 15 days to cool to ambient temperature. The images of RhB and MB and their properties are shown in Figure 1 and Table 1, respectively.\n\nThe size of each experimental plot was 20 m2 (4 m × 5 m) which were arranged in a randomized complete block design with three replications. Each plot was separated by soil banks and covered with mulch film. Five treatments with RhB and MB incorporated into the soil paddy field comprised 0 t ha−1 (conventional rice cultivation without biochar supplementation), 5 t ha−1, and 10 t ha−1 (based dried weight) named CT0, RhB5, RhB10, MB5, and MB10, respectively. Biochar was manually spread on the soil surface of each pot and evenly incorporated into the plow layer of soil (approximately 20 cm) by shovels and rakes before sowing. Biochar additions were applied one time solely at the beginning of the experiment.\n\nAccording to the local crop calendar, the experiment time corresponded with the Spring-Summer (SS) season (the second crop) (Table 2). This is a transitional season between the dry and wet seasons. Rice straw and rice stubble from the previous rice crop cycle (Winter-Spring) were plowed by a hand tractor and underwent a 7-day fallow period before sowing. A short-duration variety of rice (IR50404 cultivar) typically grown in VMD was used in this field experiment (85-90 days of maturity). Pre-germinated seeds were sown on the wet-levelled soil using drum seeders at a rate equivalent to 120 kg ha−1. The irrigation followed regionally typical water management based on the farmer’s practical experience.\n\n1) dd/mm/yyyy.\n\n2) The numbers in parenthesis indicate the amount (kg ha−1) of fertilizers applied in terms of N, P and K, respectively.\n\nInorganic fertilizers with the total amount of 80 kg N ha−1, 40 kg P2O5 ha−1 and 40 kg KCl ha−1 were applied. The fertilization was divided into intervals at 9, 23, and 38 days after sowing (DAS) by broadcasting. Nitrogen (N) was applied as urea at a rate of 16-32-32 kg N ha−1 (broadcasted three times). Phosphorus (P) was applied as superphosphate at a rate of 8-16-16 kg P2O5 ha−1 tolerant (broadcasted three times). Whereas potassium (K) was applied as potassium chloride at a 20-0-20 kg KCl ha−1 rate (broadcasted twice). The rice cropping calendar and fertilizer application are shown in Table 2.\n\nScanning electron microscope (SEM) images of RhB and MB were captured by microscope (TM-1000, Hitachi, Japan). Specific surface area and total pore volume were determined using BET Surface Area Analyzer (Quatachrome Nova 1000e, USA).\n\nA weather station (WS-GP1, Delta-T Devices, Cambridge, UK) was installed on-site to record hourly temperature and rainfall at the experimental site. Redox potential (Eh) at plow-layer soil (20 cm) was measured by using platinum-tipped electrodes pined into the ground at a depth of 5, 10, and 20 cm; a portable Eh meter (HM31P; TOA-DKK, Japan) was connected to the electrodes to record soil Eh values at corresponding times to gas sampling. Surface water levels were also recorded simultaneously with gas sampling, using a ruler to read values directly in a plastic-perforated tube pre-installed in each plot.\n\nTopsoil samples (0-20 cm) in each plot were collected before adding biochar and harvest by an auger 3 cm diameter. Visible remaining biomass was eliminated before air drying and sieved at 2.0 mm. Initial soil samples (n = 15) were mixed into a collective sample for analysis. Harvest soil samples were collected for each plot separately. Physical soil properties were measured as follows: soil texture - Pipette Robinson method (Carter and Gregorich, 2008), bulk density - Core method, and the particle density of soil (Blake and Hartge, 1986). Biochar and soil chemical properties were detected as follows: pH (H2O) – a portable pH meter (HANA, Germany), soil organic matter (SOM) and total organic C (TOC) – Walkley and Black (1934), total P - Bowman (1988), available P (AP) - Olsen and Sommers (1982), total N – semi-micro Kjeldahl method (Bremner, 1996), anaerobically mineralized N (AN)– a 7-day anaerobic incubation at 40 °C (Keeney and Bremner, 1996), and CEC and exchangeable cations – Thomas (1982).\n\nRice yield was determined by harvesting from a 2.5 m × 2.0 m area in each plot at physiological maturity and removed unfilled grains by water before sun drying. A grain moisture tester (Riceter f2, Kett Electric Laboratory, Tokyo, Japan) was used to measure moisture content. The presented rice yield was adjusted to a 14% moisture content.\n\nThe closed chamber method was used to collect gas samples. A chamber was made from transparent polyvinyl chloride (PVC) panels with a 1.5 mm thickness. The cross-sectional area was 0.25 m2 (0.5 m × 0.5 m). The height of the chamber was 70 cm from the bottom to the top layer. The chamber inside was equipped with a circulating fan, a temperature meter, and a pressure control plastic bag as described in detail by Minamikawa (2015). The chamber was placed on a plastic pre-installed base (a groove 4.5 cm depth) in each plot and sealing off by water before sampling. After chamber closure, a syringe (50 mL) was utilized to take inside gas at 1, 11, 21, and 31 minutes. Then, gas samples were injected into a 20-mL evacuated vial. The gas sampling was carried out from 10 DAS to 73 DAS at 7-day intervals. The concentrations of CH4 and N2O were analyzed with a gas chromatograph (8610C, SRI Instruments, CA, USA) equipped with a flame ionization detector (FID) and an electron capture detector (ECD) for the analysis of CH4 and N2O, respectively. The columns for the analysis of CH4 and N2O were packed with Porapak Q (50–80 mesh); dinitrogen (N2) was used as the carrier gas for both FID and ECD.\n\nPorosity was calculated by dividing volume pores (based on the subtraction between bulk density and particle density of soils) by volume total (Flint and Flint, 2002). CH4 and N2O fluxes were calculated by a linear progression of gas concentration change over time, and total fluxes of CH4 and N2O were calculated using a trapezoidal integration method described by Minamikawa (2015). Global warming potential (GWP) was calculated based on CO2 equivalence (1 CH4 = 34 CO2-eq; 1 N2O = 298 CO2-eq) at a 100-year scale of climate-carbon feedbacks (Myhre et al., 2013). Yield-scale GWP was calculated by dividing the GWP by grain yield (Minamikawa et al., 2021).\n\nOne-Way analysis of variance (ANOVA) was used to assess the effects of each biochar on grain yield, gas fluxes, GWP, yield-scale GWP, and soil improvement. The difference of treatments was carried out using Duncan’s method for all pairwise multiple comparison procedures. Linear regression analyses were performed to assess the relationship between Eh change and methane emission. We also analyzed the relationship between biochar application rate and gas emissions. In the statistical analysis, we did not compare the difference between RhB and MB. All analyses were carried out using R stats Version 4.2.0 (R Project for Statistical Computing, RRID:SCR_001905). The results are presented in tabular form with the values including mean ± standard deviation (SD) and the different symbols with a confidence level of 95%.\n\n\nResults\n\nThe mean air temperature and the total rainfall during the experiment were 28.9 °C and 429 mm, respectively (Figure 2). High rainfall was observed between –40-60 and –65-80 DAS. Figure 3 shows that the flooding water regime was predominantly observed during the experimental regression. The trend of water levels variation was similar over treatments. Water was irrigated from 7 DAS, reflooded 3-5 cm from soil surface for fertilizing (9, 23, and 38 DAS) and respective multiple drainage practice (−10 to 5 cm) (Uno et al., 2021) was carried out for the remaining periods. Fifteen days before harvesting (70 DAS), the soil was drained and kept saturated to minimize rice lodging and easy-to-harvest grain. Rice plants flowered and headed during 45-60 DAS.\n\nError bars indicate the standard error (n = 3). Vertical dotted lines illustrate agronomic management of the first, the second and the third topdressing fertilizer (F1, F2 and F3, respectively), drainage (D) and harvest (H).\n\nCH4 emissions gradually increased in the early rice growth stage (0-17 DAS) and almost stopped after drainage (70 DAS) (Figure 3). It should be noted that CH4 flux was predominant in the period from 17 – 59 DAS and several CH4 flux peaks were observed between treatments (i.e., three peaks were observed in MB5 and MB10). Maximum CH4 flux peaks reached simultaneously in all treatments after 31 DAS. Highest peaks between treatments are represented in a descending way as follows: CT0 > MB5 > MB10 > RhB5 > RhB10. Compared to the CT0 treatment, biochar application reduced total CH4 emissions significantly (Table 3). Particularly, RhB5 and RhB10 mitigated total CH4 flux from 24.2 to 28.0%, respectively, while MB5 and MB10 alleviated between 22.0 and 14.1%, respectively. Irrespective of RhB and MB, the CH4 flux was insignificant with an increasing biochar addition rate from 5 to 10 t ha−1 (P < 0.01). There was a negative linear regression relationship between biochar application rate and total CH4 emission (P < 0.001, r2 = 0.825) (Figure 4). In contrast, the linear regression of melaleuca biochar was poorly explained with increasing biochar amendment rate and total CH4 flux (P = 0.095, r2 = 0.254).\n\n1) Data represent as means ± SD (n = 3).\n\n2) CT0, control treatment; RhB5 and RhB10, 5 and 10 t ha−1 rice-husk biochar amendment, respectively; MB5 and MB10, 5 and 10 t ha−1 melaleuca biochar amendment, respectively.\n\n3) Statistical analysis did not compare between RhB and MB. The letters indicate significant difference according to Duncan’s multiple range test (*P < 0.001, P < 0.01, *P < 0.05 and †P > 0.05). Normal and capital lowercases indicate a significant difference between CT0 vs. RhB and CT0 vs. MB, respectively.\n\nEach symbol represents one replication in each treatment.\n\nN2O was released mainly in the early stage of rice growth in all treatments (Figure 3). The highest N2O flux peaks were observed in the CT0 (24 DAS). All measured values were below 1.5 mg N2O m−2 h−1. As observed, N2O flux flushed mainly during the fertilizing period from 9 to 38 DAS, even though experimental pots were predominantly flooded, especially in the CT0 accounted for 56.8% in total, while RhB and MB varied by 50.6-53.1% and 52.3-47.6%, respectively. Total N2O emission was reduced in RhB or MB applied soil compared to CT0 (Table 3). Specifically, RhB10 significantly reduced by approximately 41.0%, whereas MB5 and MB10 by 38.5 and 56.4%, respectively. However, the reduction of total N2O flux was insignificant in MB5. As a result, there were different negative linear relationships of biochar application rate and total N2O flux (RhB, P = 0.012, r2 = 0.619; MB, P = 0.002, r2 = 0.757) (Figure 4).\n\nBiochar addition to the soil slightly increased rice yield compared to the CT0, but the statistical analysis was insignificant (Table 3). A similar pattern about emissions was seen among GWP, yield-scaled GWP, and total CH4 flux due to CH4 flux was greatest contribute to GWP, yield-scaled GWP. The RhB additions significantly decreased the GWP and yield-scaled GWP by 24.4 – 30.0% and 25.8 – 31.8% for RhB5 and RhB10, respectively. Although MB significantly diminished the GWP and yield-scaled GWP by 24.8 – 21.09% and 27.8 – 24.5%, respectively, there was no significant difference between MB5 and MB10.\n\nA similar performance pattern of soil Eh condition was seen among treatments (Figure 3). Eh reduced after initial irrigation and was seen to reach a stable level (below -250 mV) during the rice growth period from 17 to 66 DAS. Whereas the final drainage rapidly increased the soil Eh condition (73 DAS) in all treatments. The supplementation of RhB and MB obviously improved soil Eh condition compared to the CT0 by 7.44 – 14.5% and 10.7 – 19.0%, respectively (Table 4). There was a negative linear relationship between hourly CH4 flux and the Eh values in RhB (P < 0.001; r2 = 0.552) and MB (P < 0.001; r2 = 0.502) (Figure 5).\n\n1) Data represent as means ± SD (n = 3).\n\n2) Abbreviations are the same as Table 3.\n\n3) Mean value is based on the whole values measured during the experimentation in each plot at 3 soil levels depth comprising 5, 10 and 20 cm between 10 and 64 DAS.\n\n4) Statistical analysis was carried out as the same as Table 3.\n\nTable 4 represents the soil characteristic differences between treatments at the time of harvest. Overall, although biochar amendment was seen to increase soil pH slightly, statistical analysis implied no significant difference between treatments. Yet, biochar amendment significantly reduced the soil bulk density (RhB5, 19%; RhB10, 23%; MB5, 22.7% and MB10 26.8%) and ameliorated the soil porosity (RhB5, 8.2%; RhB10, 11.8%; MB5, 2.2%, and MB10 9.6%). However, increasing RhB and MB biochar application rate from 5 to 10 t ha−1 did not significantly change soil bulk density and porosity. Moreover, intensifying biochar incorporation significantly increased SOM by 38.6 – 52.7% for RhB and 25.4 – 45.9% for MB. Notably, AN in biochar-applied treatments was higher than that of the CT0 by 44.8 – 38.3% and 35.5 – 55.1% for RhB and MB, respectively. AP significantly increased in the MB treatments by 32.1 – 51.58% but did not in RhB. Although additional biochar increased the available and mineralized nutrients, statistical analysis results showed no significant difference between biochar application rates of 5 to 10 t ha−1 (Table 4) (Tran Sy et al., 2021).\n\n\nDiscussion\n\nConventional practices without biochar application released 18.6 g CH4 m−2 and 0.39 g N2O m−2 (Table 3). These values are in accordance with previous findings conducted in the VMD (Vo et al. 2020; Minamikawa et al. 2021; Uno et al. 2021). Notably, RhB and MB amendments under typically local water management, and conventional practices significantly reduced CH4 flux by 24.2 % in RhB5, 28.0 % in RhB10, 22.0 % in MB5, 14.1 % in MB10 and N2O flux by 38.5 % in RhB5, 56.4 % in RhB10, 25.6 % in MB5, 41.0 % in MB10, and slightly improved rice yield (2.41-4.21%) (Table 3). Similarly, Yang et al. (2019) demonstrated that biochar additions (20 - 40 t ha−1) under controlled irrigation in the Taihu Lake region, China mitigated both CH4 and N2O emissions by 35.7% and 21.5%, respectively, and simultaneously enhanced rice yield by 16.7-24.3%. Moreover, Wu et al. (2019b) reported that biochar additions (20 - 40 t ha−1) significantly decreased CH4 and N2O fluxes by 11.2-17.5% and 19.5-26.3%, respectively, and increased grain yield by 7.9-9.2%. In line with our findings, a long-term biochar application (5 – 10 t ha−1) in China's typical double rice plantation region also significantly decreased CH4 flux by 26.18% (Qin et al. 2016). Nevertheless, Wang et al. (2011) reported that the biochar incorporation (50 ton ha−1) into the soil significantly decreased N2O flux by 41.4-93.5% in lab-scale experiments. In parallel, a meta-analysis based on 30 studies with 261 experimental treatments (lab-scale and pilot-scale) from 2007 to 2013 demonstrated that the addition of biochar reduced N2O emissions by 54% (Cayuela et al. 2014). However, Koyama et al. (2015) reported that biochar application (10-40 t ha−1) reduced CH4 flux but did not N2O. In the case of Liu et al. (2014), biochar supplementation (24-48 t ha−1) significantly reduced CH4 flux by 33.9-40.2%, while N2O flux significantly increased by 150 to 190%. Overall, biochar amendment could reduce CH4 flux from a rice paddy field, but in some cases, the effect on N2O flux remains uncertain. Our study demonstrated that rice husk and melaleuca biochar applications with a range of 5-10 t ha−1 significantly reduced both CH4 and N2O fluxes within a Thionic Glycesol soil in the VMD. Albeit, the biochar application rate between 5 and 10 t ha−1 hardly obtained the disparity of CH4 and N2O emissions. Thus, a wide range of biochar application amounts should be evaluated to provide more tailored recommendations.\n\nThe CH4 mitigation by biochar application consistently pertains to the increasing soil oxidation rate and methanotrophs community. Although we did not determine the number of methanogens and methanotrophs, Nan et al. (2021) demonstrated that biochar application stimulates the abundance in either methanogens or methanotrophs, with a high amount of methanotrophs detected in most cases resulted in decreasing of CH4 flux. Moreover, Wu et al. (2019a) reported that biochar applications to fertilized paddy field soils increased the total type I pmoA (preferred the CH4 environment) and type II pmoA (more dynamic in low CH4 conditions) methanotrophs comparing to non-amended biochar, indicating that CH4 flux mitigation by promoting potential CH4 oxidation. Thus, we adopted a hypothesis that the balance of activities between methanogens and methanotrophs in a site-specific environment results in either an increasing or decreasing CH4 flux. Feng et al. (2012) revealed the main mechanisms of CH4 flux reduction in a biochar-supplemented field were by (1) increased methanotrophic proteobacterial abundance significantly and (2) decreased the methanogenic to methanotrophic proportion substantially. Thus, an increase of methanotrophs dynamic in paddy field soil by biochar addition can be expected to play a vital role in mitigating CH4 fluxes. Our study demonstrated that rice husk and melaleuca biochar could promote low-GHG emissions in the rice production system in the VMD.\n\nWe achieved N2O flux reduction by incorporating biochar into the topsoil layer when compared to the non-amended biochar field. However, several hypotheses supposed that soil applied with biochar could not decrease the N2O flux (Koyama et al., 2015; van Zwieten et al., 2010). Similar to our field study, several findings achieved a total N2O flux reduction (Shaukat et al., 2019; Zhang et al., 2010). The mitigation of N2O flux in biochar-treated soils could be attributed to soil moisture contents and nitrification processes (Ameloot et al., 2016). In agreement with the hypothesis, Shaukat et al. (2019) demonstrated that fields with biochar added retained 9-14% higher moisture contents than fields without biochar amended and resulted in a significant reduction of the N2O flux. Supporting the idea, Wang et al. (2013) revealed the relationship between the denitrifying community and N2O flux change, where biochar supplementation significantly shifted the abundance of NO3-utilizing bacteria (carrying the nirK and nirS genes), leading to less N2O generation and more N2O-consuming bacteria (carrying the nosZ gene). Moreover, Cayuela et al. (2013) used 15N gas-flux to observe the reduction of N2O/(N2+N2O) and demonstrated that biochar facilitated the last step of denitrification. The key mechanisms of N2O flux reduction under biochar amendment were by (i) stimulated nitrification generation via electron donation, a decrease in total denitrification by serving as an alternative electron acceptor by acting as electron shuttle to soil NO3− consuming microorganisms (Cayuela et al., 2013), and (ii) based on the entrapment of N2O in water-saturated soil pores and co-occurrent stimulation of microbial N2O reduction deriving in an overall decrease of the N2O/(N2O + N2) ratio (Harter et al., 2016). Therefore, biochar could be attributed as a decisive factor to inhibit N2O production and simultaneously stimulate N2O utilization. As such, these findings and the above-discussed mechanisms strongly support our findings in suggesting N2O flux reduction from biochar amendment in the rice paddy field.\n\nOur study showed that N2O emission was mainly concentrated during fertilization, which indicates fertilization provides more available N driving for soil N2O emission. Xie et al. (2013) observed 15N abundance significantly intensified by the application of 15N-enriched urea. Our study did not measure NH4+ or NO3− concentration during fertilizing, so the mechanism remains uncertain. N2O emission via the nitrification process directly pertains to soil physical, chemical, and biological properties (Huang et al., 2019). Thus, we speculate that N fertilizing increased the nitrification activities and stimulated the strong metabolism of potential N2O-producing bacteria. Minamikawa et al. (2021) reported that higher N availability levels in soil than rice plant uptake demands resulted in increasing N2O emissions. Although N-fertilizing obviously promoted N2O emissions for the majority of time, N2O emission peaks of biochar-amended soil were lower than that of biochar-unamended soil. This would indicate that biochar potentially changed the functionality and diversity of denitrifiers within the soils and inhibited the conversion of NO2− and NO3− to N2O (Zhang et al., 2010).\n\nWater management is a crucial factor in the strategy of GHGs reduction, although we achieved the GHGs reduction under typical water management when most of the time the soil was flooded. Multiple-flooded times in this study were due to the combination of high rainfall in the transition season (rainfall, Figure 2; water level, Figure 3) and the typical flooding water management practice of the farmers in the region. Uno et al. (2021) conducted a 2-year field experiment in An Giang province in the VMD and demonstrated that AWD (known as multiple drainages) significantly reduced CH4 by 35%, while found no difference in N2O emissions, but a 22% yield improved. Moreover, Minamikawa et al. (2021) registered that the intermittent irrigation technique is also a promising approach to mitigate CH4 emissions by reductive soil conditions. Thus, integrating AWD and intermittent irrigation by incorporating biochar into the soil under the MD’s edaphology, climate, and traditional practices could be feasible for further works.\n\nThere is a negative correlation between CH4 flux and RhB application rate (P < 0.001, r2 = 0.825) (Figure 4). It is indicated that CH4 flux decreased with the increase of rice-husk biochar application (Xiao et al. 2018). On the other hand, although increasing MB application rate could mitigate the CH4 emission, the relationship found a poor explanation (P = 0.095, r2 = 0.254). This contrast could be partly attributed to biochar-carbonized properties. MB was low in the specific surface area and total pore volume compared to RhB (Table 1). Ji et al. (2020) revealed that biochar structure intimately related to anaerobic CH4 oxidation and created a suitable environment for CH4-consuming bacteria.\n\nSimilarly, we found a negative correlation between the N2O flux reductions and the application rate of RhB (P = 0.012, r2 = 0.619) and MB (P = 0.002, r2 = 0.757). In agreement with our finding, a meta-analysis of Cayuela et al. (2014) showed a negative relationship between biochar application rates and reduced N2O flux, where sufficient N2O reduction was 1-2% biochar amendments, whereas, incorporating more than 10% of biochar into the soil was found to reach up to 80%. In line with our study, Huang et al. (2019) also showed a negative relationship between biochar application rates and N2O flux. Overall, the increase of biochar application rates could potentially stimulate the CH4 and N2O reduction. However, for CH4 and N2O fluxes, the application of 5 and 10 t ha−1 remains unclear.\n\nOur study found that the negative linear relationship between soil Eh and hourly CH4 flux with RhB (P < 0.001; r2 = 0.552) and MB (P < 0.001; r2 = 0.502) (Figure 5). Similar results were also observed by Wang et al. (2018). This indicates that an increase of soil redox potential decreased CH4 emission, which is in line with the report by Towprayoon (2020). Moreover, soil Eh remained below −250 from 17 to 66 DAS in our study (Figure 4), implying a favorable condition for CH4 emission (Wang et al. 1993). Final drainage rapidly increased soil Eh and reduced CH4 flux (Figure 3), indicating the strong sensibility of soil Eh and CH4 flux under water management.\n\nBiochar application increased soil Eh compared to non-amended soils (Table 4). This indicates that biochar was the critical factor contributing to the positive effects of anaerobic CH4 oxidation activities known as the electronic accepting capacities (EAC) of biochar (Nan et al., 2021). The supplementation of biochar intensifies oxygen-containing functional groups (carboxyl, carbonyl, quinone phenolic hydroxyl group) and positively improves biochar redox potential (Klüpfel et al. 2014; Wu et al. 2016). The increase of Eh and the reduction of CH4 emissions could also be explained by the porosity and absorbability characteristics of biochar, which enable robust CH4-utilizing bacteria activities and intensify the diffusion and metabolism process. In a similar way, biochar incorporation into soils improves soil aeration, creating a favorable environment for methanotrophic bacteria resulting in soil Eh amelioration and better reduction of CH4 oxidation (Feng et al., 2012).\n\nAlthough biochar amendments could improve yield (2.41-4.21%) (Table 3), multiple comparison analyses found no significant difference between amended and unamended soils. Several studies have found similar results (Qin et al. 2016; Nguyen et al. 2016). The undistinctive grain yield could be partly attributed to spatial and temporal variations, i.e., climatic conditions, field practices, soil substrates (Xie et al. 2013).\n\nBiochar-amended soil significantly decreased GWP by 21.1-30.0% and yield-scaled GWP by 24.5% - 31.8% (Table 3). It was indicated that RhB and MB application potentially mitigates total CH4 and N2O emissions without scarifying grain yield. Yang et al. (2019) performed a double-season field experiment on biochar applications ranging from 20 to 40 t ha−1 and found that the average GWP and yield-scaled GWP reduced by 18.7% - 16.4%, and 80.3% - 41.6%, respectively. Similarly, Zhang et al. (2019) observed a six-year field experiment on biochar-applied soils at rates of between 20-40 t ha−1 and showed a GWP and yield-scaled GWP reduction by 12.1-18.4% and 35.9-56.4%, respectively. Here we observed that CH4 flux was the key contributor in the GWP and yield-scaled GWP via the field experiment in the VMD’s transition season, while N2O flux was more neglectable. Thus, future works should emphasize on reducing the GWP, yield-scaled GWP, and concentrate on the CH4 mitigation technology solutions rather than N2O emissions.\n\nSoil improvement under short-term and long-term biochar applications has been widely recognized. Our study showed that biochar amendment insignificantly increased soil pH (Table 4), which indicated no effect of biochar addition on soil pH perfection as suggested by previous studies (Yang et al., 2019). However, biochar amendment significantly decreased the soil bulk density and improved soil porosity in comparison to non-amended soils. Furthermore, higher applied biochar rates showed lesser soil bulk density and higher porosity indicating that biochar directly upgraded soil physiology. Amelioration of soil surface area and porosity by biochar amendment intensifies soil aeration and functions of aeration, such as CH4 oxidation, and provides habitat for methanotrophs (Nan et al., 2021). Moreover, it stimulates NH4+ absorbance ability resulting in suppressing nitrification processes and N2O flux reduction in the field (Wang et al., 2020).\n\nIt is evident that increasing biochar application boosted SOM and AN, with a slightly increased available P through the season (Table 4). The increasing of SOM and AN showed a high nutrient availability in the soil. Notably, the soil improvement did not increase soil CH4 and N2O emissions as above-mentioned and discussed. AN could be used as a soil health indicator (García et al., 2020). The interdependence among AN, SOC, and particulate OC was demonstrated by a positive correlation (Domínguez et al., 2016). In connection with our study, Yang et al. (2019) observed that biochar amendment slightly increased SOC, significantly increased NH4+ by 47.7%, and significantly decreased NO3− by 30.4%. Incorporating biochar into soils could inhibit nitrification and produce more NH4+ than NO3− consisting of an anoxic environment (water level and redox potential; Figure 2). Increasing NH4+ concentrations and declining NO3− concentrations would partly explain the enhanced CH4-consuming figure and N2O oxidation (Xiao et al., 2018). Overall, biochar application offers benefits not only for nutrients availability, but also for GHGs mitigation.\n\n\nConclusions\n\nThis study assessed the effects of rice husk biochar or melaleuca biochar amendment at 5 or 10 t ha−1 on CH4 and N2O emissions and the physiochemical soil properties after rice cultivation under typical water management and conventional practice regime in the VMD. Incorporating biochar into soils significantly mitigated CH4 and N2O emissions without reducing grain yield. Consequently, a lower GWP and yield-scaled GWP from biochar-amended soils were achieved. Although higher biochar applications decreased CH4 and N2O emissions, there was no significant difference between biochar-amended rates. Biochar significantly increased soil Eh conditions. There was a negative linear relationship between soil Eh and CH4 emission rate for biochar-applied fields. N2O emissions from biochar fields were relatively low and mostly concentrated during the fertilization period. Biochar amendments improved soil fertility via physical properties of soils by decreasing bulk density and intensifying porosity and the chemical characteristics of the soils by ameliorating SOM, AN and AP, but did not affect soil pH. Similar to GHG emissions, biochar application rates of between 5 and 10 t ha−1 could not obtain significant soil improvement. This study will help lower-GHG emissions from rice farming practices in the VMD. Further works should study the combination of biochar-application rates and effective water irrigation techniques on different soils in the VMD.\n\n\nData availability\n\nFigshare: Biochar reduces GHGs from paddy fields. https://doi.org/10.6084/m9.figshare.16625137.v1 (Tran Sy et al., 2021).\n\nThis project contains the following underlying data:\n\n- Nam et al_Raw data biochar_F1000research.xlsx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThis study is funded in part by the Can Tho University Improvement Project VN14-P6, supported by a Japanese ODA loan. We would like to thank Cuu Long Delta Rice Research Institute (CLRRI), Vietnam, and Mr. Ho Minh Nhut (master student K25 in Can Tho University, Vietnam) for their support of this study. We also thank Dr. Nigel Downes for proofreading the manuscript\n\n\nReferences\n\nAmeloot N, Maenhout P, De Neve S, et al.: Biochar-induced N2O emission reductions after field incorporation in a loam soil. Geoderma 2016; 267: 10–16. Publisher Full Text\n\nBlake GR, Hartge KH: Bulk density and particle density. ‘Methods of soil analysis. Part 1’. Agronomy Monograph 9 Klute A, editors. Madison, WI: ASA and SSSA; 1986; pp. 363–382.\n\nBowman RA: A rapid method to determine total phosphorus in soils. Soil Sci. Soc. Am. J. 1988; 52: 1301–1304. Publisher Full Text\n\nBremner JM: Nitrogen-Total. Methods of Soil Analysis: Part 3 Chemical Methods, 5.3; SSSA Book Series Sparks D, Page A, Helmke P, et al., editors. Madison, WI, USA: Wiley & Sons; 1996; pp. 1085–1121. 9780891188667.\n\nBurrell LD, Zehetner F, Rampazzo N, et al.: Long-term effects of biochar on soil physical properties. Geoderma. 2016; 282: 96–102. 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Publisher Full Text\n\nZhang A, Cui L, Pan G, et al.: Effect of biochar amendment on yield and methane and nitrous oxide emissions from a rice paddy from Tai Lake plain. China. Agric. Ecosyst. Environ. 2010; 139: 469–475. Publisher Full Text" }	[ { "id": "99426", "date": "04 Jan 2022", "name": "Azeem Tariq", "expertise": [ "Reviewer Expertise Climate change mitigation", "GHG emissions", "sustainable crop rotations", "ecosystem modeling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of \"Rice husk and melaleuca biochar additions reduce soil CH4 and N2O emissions and increase soil organic matter and nutrient availability\" present an effort in assessing the effects of two biochar amendments on soil CH4 and N2O emissions and effects on soil physiochemical properties based on 80 days of field study. The overall paper has been written in a good English and in a logical manner. However, there are some minor issues that authors need to deal before considering this paper for indexing.\nAuthors used the term “soil organic matter and nutrient availability” in title, but they have focused on different soil properties e.g. soil porosity, soil bulk density, soil redox potential, soil organic matter and nitrogen. Authors should use the term soil physiochemical properties instead.\nAuthors did not explain the results related to soil properties in the abstract section (e.g. there is no explanation about soil organic matter and nitrogen contents in abstract).\nAuthors stated that they used 95 % confidence level, but the result section predicted that authors have used different levels of probability for difference analysis. Author should explain the analysis carefully in the statistical analysis section in Material and Methods.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7791", "date": "15 Feb 2022", "name": "Thao Huynh Van", "role": "Author Response", "response": "Thank you very much for giving us constructive comments that helped us improve this paper immensely. All your comments have been carefully read and revised to elucidate underlying aspects. Our responses to your comments are as follows: Comment 1: Authors used the term “soil organic matter and nutrient availability” in title, but they have focused on different soil properties e.g. soil porosity, soil bulk density, soil redox potential, soil organic matter and nitrogen. Authors should use the term soil physiochemical properties instead. Reply 1#: Thank you very much for the recommendation for the title term of “soil physiochemical properties”. The recommendation has been adopted. The revision is as follows “Rice husk and melaleuca biochar additions reduce soil CH4 and N2O emissions and increase soil physicochemical properties.” Comment 2: Authors did not explain the results related to soil properties in the abstract section (e.g. there is no explanation about soil organic matter and nitrogen contents in abstract). Reply 2#: We revised the abstract with additional information related to the explanation and implication for improving soil physiochemical characteristics. The revision is as follows “The results showed…Ameliorating soil aeration and functions by adding RhB and MB resulted in improving soil physicochemical characteristics, especially significant SOM and AN boosting, which indicate better soil health, structure, and fertility”. Comment 3: Authors stated that they used 95% confidence level, but the result section predicted that authors have used different levels of probability for difference analysis. Author should explain the analysis carefully in the statistical analysis section in Material and Methods. Reply 3#: We added more information to the statistical level to make it clearer to readers. The supplementations are as follows “The results are…Significant different comparison among treatments was considered at Duncan’s multiple range test (*P < 0.001, P < 0.01, P < 0.05 and †P > 0.05)” We look forward to hearing from you. Best regards, Authors." } ] }, { "id": "120157", "date": "08 Feb 2022", "name": "Bui Truong Tho", "expertise": [ "Reviewer Expertise Hi-tech Application in Agriculture", "Climate change mitigation", "GHG emissions", "Sustainable crop rotations", "Plant ecophysiology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, \"Rice husk and Melaleuca biochar additions reduce soil CH4 and N2O emissions and increase soil organic matter and nutrient availability\" aims to evaluate the impacts of two biochar amendments on soil CH4 and N2O emissions and its effect on soil physiochemical properties in Mekong Delta based on 80 days of field study.\nThe topic is interesting and poorly treated in literature. The data are correctly processed and the results are well discussed. However, there are some minor issues and my reviews are focused on improving a revision of the current article, and I hope the authors can accommodate my suggestions.\nSite description: “air temperature (27.5 – 27.5 °C)” I think there is a mistake, authors should double-check the information.\n\nStatistical analysis: Authors should confirm that all data were test homogeneity of variance for using One-Way analysis of variance (ANOVA).\n\nAuthors used “Vietnamese Mekong Delta (VMD)”, some places used “Mekong Delta (MD)”, it should unified in all the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7792", "date": "15 Feb 2022", "name": "Thao Huynh Van", "role": "Author Response", "response": "Thank you very much for your constructive comments. All your comments have been carefully read and revised by the authors. Our responses are enclosed as follows: Comment 1#. Site description: “air temperature (27.5 – 27.5 °C)” I think there is a mistake, authors should double-check the information. Reply 1: Thank you for finding this mistake. We re-referred to the original document and corrected the information in the manuscript. The air temperature should be 27.5oC instead. Comment 2#: Statistical analysis: Authors should confirm that all data were test homogeneity of variance for using One-Way analysis of variance (ANOVA). Reply 2: We amended additional information related to the homogeneity of variance in the statistical analysis. The revised is as follows “Significant different comparison among treatments was considered at Duncan’s multiple range test (P < 0.001, P < 0.01, *P < 0.05 and †P > 0.05) after passing homogeneity of variance”. Comment 3: Authors used “Vietnamese Mekong Delta (VMD)”, some places used “Mekong Delta (MD)”, it should be unified in all the manuscript. Reply 3: We have unified the abbreviation in all parts of the manuscript which is “VMD”. Best regards, Authors." } ] } ]	1	https://f1000research.com/articles/10-1128
https://f1000research.com/articles/11-187/v1	15 Feb 22	{ "type": "Research Article", "title": "Correlation between serum methotrexate-polyglutamate 3 (MTX-PG3) level and disease activity in rheumatoid arthritis patients: A prospective cohort study", "authors": [ "Eva Musdalita", "Rudy Hidayat", "Sumariyono Sumariyono", "Suryo Anggoro Kusumo Wibowo", "Anna Ariane", "Hamzah Shatri", "Iris Rengganis", "Dono Antono", "Rudy Hidayat", "Sumariyono Sumariyono", "Suryo Anggoro Kusumo Wibowo", "Anna Ariane", "Hamzah Shatri", "Iris Rengganis", "Dono Antono" ], "abstract": "Background: Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, characterized by systemic inflammation, joint destruction and disability. Methotrexate (MTX) is used as the primary treatment for RA patients. However, the response to MTX therapy is highly varied and difficult to predict. This study sought to determine the role of MTX by measuring the MTX polyglutamate 3 (MTX-PG3) levels and the disease activity score 28 based on C-reactive protein (DAS28-CRP) of RA patients. Method: A prospective cohort study was conducted at the Rheumatology Polyclinic of Dr. Cipto Mangunkusumo General Hospital. Thirty-four patients with RA were included and followed up to 12 weeks. The RA patients were treated with MTX 10 mg per week and an increased dose of 5 mg per week every month. DAS28-CRP and MTX-PG3 level were assessed at week 8 and 12. Multivariate logistic regression analysis was used to determine the correlation between MTX-PG3 and DAS28-CRP. Result: A total of 34 RA patients were followed and the MTX was well tolerated in which no increase of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and glomerular filtration rate (GFR) were observed. The mean scores of DAS28-CRP decreased following the MTX-treatment: 3.93, 3.22 and 2.82 at week 0, 8 and 12, respectively. In contrast, the median concentration of MTX-PG3 increased from week 8 to week 12 followed by increasing the dose of MTX. Our analysis suggested there was a moderate positive correlation between MTX-PG3 levels and DAS28-CRP score at week 8 and week 12 post-MTX treatment. Conclusion: The level of MTX-PG3 is correlated with DAS28-CRP score suggesting that MTX-PG3 could be used as an indicator to assess the disease activity in RA patients. Nevertheless, a prospective study with a higher number of patients is needed to confirm this finding.", "keywords": [ "Rheumatoid arthritis", "MTX-PG3 level", "disease activity", "DAS28-CRP", "methotrexate" ], "content": "Introduction\n\nMethotrexate (MTX) is the first-line drug to treat rheumatoid arthritis (RA) which provides higher survival rate than other disease modifying arthritis rheumatoid drugs (DMARD) and is recommended by the European League Against Rheumatism and the American College of Rheumatology. MTX has become a commonly used treatment option because it is a cost-effective and has acceptable safety profile.1–3 Although MTX is the main RA therapy and most patients show symptomatic improvement and have acceptable side effects, the response to MTX therapy is highly varied and difficult to predict.4\n\nMTX is transported into cells via the reduced folate carrier pathway and activated by polyglutamate synthase to methotrexate polyglutamate (MTX-PG).5 MTX-PG then inhibits the target enzymes such as thymidylate synthetase, dihydrofolate reductase and key enzymes for de novo purine synthesis pathway.6 Depending on the number of conjugated glutamates, MTX-PG may be present as MTX-PG1-5 or the longer chain MTX-PG (MTX-PG3-5) which is considered to be more active. MTX-PG3 is the most dominant and stable type of MTX-PG and could reflect the overall polyglutamate status.7 Therapeutic effects of MTX on RA patients depends on conversion of MTX to MTX-PG and therefore intracellular measurement of MTX-PG has been proposed as an objective method of guiding MTX therapy.8\n\nStudies have been conducted to analyze the pharmacodynamics of MTX-PG, in particular MTX-PG3, since it is expected to be used to monitor the safety and effectiveness of MTX therapy in RA patients.8,9 A prospective cohort study found that MTX-PG3 and total MTX-PG levels were associated with decreased disease activity score 28 (DAS28) as measured at 3, 6, and 12 weeks. MTX-PG3 levels also positively correlated with the number of MTX dose during the treatment.9 However, studies showed different results in which the levels of MTX-PG3 had no potential as a marker of clinical improvement in RA patients, and also there was no significant relationship between MTX-PG level with the side effects and treatment response status.10,11 Therefore, the MTX-PG concentration in erythrocytes as a predictor of response to MTX therapy in RA patients is still a conflict. Besides that, the cut-off value for MTX-PG levels is different in each country since RA is determined by specific genetic factors.12 This prospective study aimed to clarify the relationship between intra erythrocytic MTX-PG3 concentration and DAS28 based on C-reactive protein (DAS28-CRP) in Indonesia. The CRP value is considered more sensitive than short-term changes in RA disease activity.13 This study was expected to provide a better understanding of the role of MTX-PG3 in the treatment of RA.\n\n\nMethods\n\nA prospective cohort study was conducted among RA patients treated at Rheumatology Polyclinic of Dr. Cipto Mangunkusumo General Hospital (RSCM) in Jakarta. The RA patient was diagnosed based on the 2010 American College of Rheumatology/European League Against Rheumatism (2010 ACR/EULAR) diagnostic criteria which is a new and more sensitive approach in diagnosing RA. The diagnostic criteria of 2010 ACR/EULAR includes joint involvement, serological test, CRP, erythrocyte sedimentation rate (ESR), and duration of symptoms.14 Before MTX treatment, DAS28-CRP was assessed, demographic and clinical data were collected including gender, age, body mass index (BMI), CRP, nonsteroidal anti-inflammatory drugs (NSAID) use, steroid use, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), glomerular filtration rate (GFR) and erythrocyte sedimentation rate (ESR). Subsequently, the patients were treated with MTX and were followed until 12 weeks post-treatment. The DAS28-CRP examination and the concentration of MTX-PG3 were re-assessed at week 8 and 12.\n\nThe patients received MTX therapy with an initial dose of 10 mg/week and an increased dose of 5 mg/week every month according to the degree of disease activity. The patients also received methylprednisolone 4 mg twice daily and omeprazole 20 mg once daily. At week 8 and 12, the venous blood was collected at least 1.5 hours after MTX administrated.\n\nThe DAS28-CRP assessment was conducted before the MTX treatment and during the follow-up of 8 and 12 weeks post-treatment. DAS28-CRP assesses 28 tender and swollen joint counts, general health and the level of CRP (mg/L) and the score was calculated based on formula that have been provided elsewhere.15 The DAS28-CRP score interpretation are as follows: <2.6: disease remission, 2.6–3.2: low disease activity, <3.2–5.1: moderate disease activity, and >5.1: high disease activity.16\n\nTo measure the MTX-PG3, the blood samples were first centrifuged for 5 min at 3000 rpm.17 The level of MTX-PG3 was measured using chromatographic analysis by injecting 10 μL of sample into WatersTM ACQUITY UPLC BEH C18 column (Waters Corporation, Milford, MA, USA) with mobile phase of 10 mM ammonium bicarbonate at pH 10, ammonium hydroxide, and methanol. The rate was maintained at 0.3 mL/min and analysis was conducted using the program of 0-0.5 min isocratic hold 5% B, 0.5-4 min linear gradient 5-40% B; 4.0-4.25 min linear gradient 4-100% B; 4.25-4.75 min isocratic 95% B; 4.75-5 min linear gradient 100-5% B; and 5-6 min 5% isocratic.\n\nDescriptive analysis was performed to provide the distribution of the data. The MTX-PG3 levels and DAS28-CRP score were presented as mean ± standard deviation if the data normally distributed otherwise as median (min-max). At univariate analysis, Spearman’s correlation test was used to assess the correlation between the potential confounding variables with DAS28-CRP. The multivariate linear regression test was used to determine the correlation between the MTX-PG3 levels and DAS28-CRP scores by controlling for confounding variables. All analyses were conducted using SPSS software version 24 (SPSS Inc., Chicago, IL, USA, RRID:SCR_002865).\n\n\nResults\n\nA total of 34 RA patients were enrolled and analyzed, and the characteristics of the patients are presented in Table 1. No patients dropped out from the follow-up. Most of the patients (94.1%, 32/34) were female with an age range of 20 to 70 years. The mean BMI was 23.29 kg/m2, indicating that most of the patients were in the ideal weight range. All patients did not smoke and had no comorbidities. Examination of SGOT, SGPT, and GFR showed normal limits with medians of 18 U/L, 18 U/L, and 102.5 mL/min/1.73m2, respectively. The median of disease duration was 8.5 months.\n\nThe mean score of DAS28-CRP before the MTX treatment was 3.93 indicating most of the patients had a moderate disease activity state, then the DAS28-CRP scores decreased to 3.22 and 2.82 at week 8 and 12, respectively. The data of MTX-PG3 levels were not normally distributed with median at week 8 and 12 being 12.63 nmol/L and 42.67 nmol/L, respectively.\n\nA linear regression was used to identify the correlation of MTX-PG3 with DAS28-CRP and the changes of DAS28-CRP score (ΔDAS28-CRP). To do this, the confounding variables were first assessed including BMI, age, GFR, MTX dose, duration of disease, methylprednisolone dose, and number of joints affected through the Spearman’s correlation test. This analysis suggested that disease duration, methylprednisolone dose, and number of joints were potential confounding variables and therefore adjusted for further analysis (Table 2).\n\nThe multivariate analysis of the role of MTX-PG3 on DAS28-CRP with a linear regression are provided in Table 3. There was a significant correlation between MTX-PG3 levels and DAS28-CRP score at week 8 and 12 post-MTX treatment after adjusted all confounding variables, r=0.517 with p=0.022 and r=0.538 with p=0.013, respectively. Our analyses also suggested that there were significant correlations between the level of MTX-PG3 and the changes of DAS28-CRP scores (ΔDAS28-CRP) at week 8 and 12 post-MTX treatment (r=0.933 and r=0.787, respectively with p<0.001 for both) (Table 3).\n\nROC analysis was conducted using ROC curve to determine how sensitive and specific the MTX-PG3 level was to predict the disease activity of RA. Data from 12 weeks post-treatment suggested that the cut-off value of MTX-PG3 level 78.4 nmol/L was able to reduce the DAS28-CRP score by more than 1.2 times. The area under the curve (AUC) was 0.9 (95% confidence interval 0.81–1, p=0.001) with a sensitivity of 75% and a specificity of 91% (Figure 1).\n\n\nDiscussion\n\nIn this study, we treated the RA patients with 10 mg MTX as an initial dose followed by an increase of 5 mg MTX/week, every month. A previous study found that treatment with an initial dose of at least 10 mg/week for the first three months with an increase of at least 20 mg/week for six months gave an excellent clinical response.18 Our data suggested that by increasing the MTX dose the MTX-PG3 level could increase by 70.4% from week 8 to week 12. In contrast, the DAS28-CRP score decreased from 3.93 (first week) to 3.93 at week 12, indicating that the disease activity changed from moderate to low degree. Based on the changes of DAS28-CRP scores, the response of the treatment was categorized as moderate response.17,19 In this study, MTX dose and MTX-PG3 levels were well tolerated since no adverse events presented at week 8 or 12, as indicated by no increase in the levels of SGOT, SGPT and GFR reported.\n\nOur data suggested that the levels of MTX-PG3 were correlated with DAS28-CRP score at week 8 and 12 post-MTX treatment. The same results were also observed between MTX-PG3 and ΔDAS28-CRP. These suggested that the higher MTX-PG3 levels the higher the score of DAS28-CRP (i.e., the better the disease activity). These results are similar to a study in Japanese patients in which there was a significant association between MTX-PG3 level and DAS28 score from week 8 to 24 post-MTX therapy.17 The decrease in DAS28-CRP may occur in the folate reduction pathway through dihydrofolate reductase (DHFR) inhibition. Reduced DHFR activity interferes with homocysteine conversion to methionine and causes inhibition of immunoglobulin and rheumatoid factor inducers.20–22\n\nFurther analysis using the ROC curve showed that MTX-PG3 level of 78.4 nmol/L at 12 weeks post-MTX treatment could predict a good response of the MTX treatment. A previous study reported that the cut-off level of MTX-PG at week 12 was more than 74 nmol/L to reduce DAS28 score and categorized as a moderate/good response.9\n\nThere are some limitations of this study. The number of the patients included in this study were relatively small and a further study with a larger sample size and longer follow-up time is required. It should be noted that the correlation of the MTX-PG3 level with DAS28-CRP score and ΔDAS28-CRP was influenced by confounding variables including duration of disease, methylprednisolone dose, and number of joints involved. Confounding factors are the main concern in the observational cohort study since no randomization was conducted.23 Nevertheless, we have tried to minimize the bias by adjusting those co-founding in the final analysis. In addition, several studies have shown that genetic factors play a role in the variability of treatment of each patient, in particular variations on gene encoding enzymes in the folate metabolism pathway.5,24 In this study, genetic factors were not controlled and could be the source of bias.\n\n\nConclusion\n\nOur study suggests a moderate positive correlation between MTX-PG3 level and DAS28-CRP score in RA patients after 8 and 12 weeks post-MTX treatment. This indicates that level of MTX-PG3 may potentially be used to predict the degree of RA disease activity and as an indicator of the patient response to MTX treatment.\n\n\nData availability\n\nFigshare: Correlation between serum methotrexate-polyglutamate 3 (MTX-PG3) level and disease activity in rheumatoid arthritis patients: A prospective cohort study https://doi.org/10.6084/m9.figshare.18079136.\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: ‘Correlation between serum methotrexate-polyglutamate 3 (MTX-PG3) level and disease activity in rheumatoid arthritis patients: A prospective cohort study’, https://doi.org/10.6084/m9.figshare.18743006.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nEthics statement\n\nThe protocol of the study was approved by the Ethics Committee of the Faculty of Medicine, Universitas Indonesia and Dr Cipto Mangunkusumo General Hospital (KET-1144/UN2.F1/ETIK/PPM.00.02/2020). All patients provided written informed consent prior to participate in this study. All works were conducted in accordance with The Code of the World Medical Association (Declaration of Helsinki).", "appendix": "Acknowledgments\n\nWe would like thanks to all patients participated in this study and all staff at Rheumatology Polyclinic of Dr Cipto Mangunkusumo General Hospital, Jakarta.\n\n\nReferences\n\nSmolen JS, Landewe R, Breedveld FC, et al.: EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update. Ann Rheum Dis. 2014 Mar; 73(3): 492–509. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFunk RS, van Haandel L , Becker ML, Leeder JS: Low-dose methotrexate results in the selective accumulation of aminoimidazole carboxamide ribotide in an erythroblastoid cell line. J Pharmacol Exp Ther. 2013 Oct; 347(1): 154–163. PubMed Abstract \| Free Full Text\n\nSingh JA, Saag KG, Bridges SL Jr., et al.: 2015 American College of Rheumatology Guideline for the Treatment of Rheumatoid Arthritis. Arthritis Rheumatol. 2016 Jan; 68(1): 1–26. PubMed Abstract \| Publisher Full Text\n\nSmolen JS, Landewe R, Bijlsma J, et al.: EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2016 update. Ann Rheum Dis. 2017 Jun; 76(6): 960–977. PubMed Abstract \| Publisher Full Text\n\nDervieux T, Zablocki R, Kremer J: Red blood cell methotrexate polyglutamates emerge as a function of dosage intensity and route of administration during pulse methotrexate therapy in rheumatoid arthritis. Rheumatology (Oxford). 2010 Dec; 49(12): 2337–2345. PubMed Abstract \| Publisher Full Text\n\nMikkelsen TS, Thorn CF, Yang JJ, et al.: PharmGKB summary: methotrexate pathway. Pharmacogenet Genomics. 2011 Oct; 21(10): 679–686. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoolf RT, West SL, Arenas-Hernandez M, et al.: Methotrexate polyglutamates as a marker of patient compliance and clinical response in psoriasis: a single-centre prospective study. Br J Dermatol. 2012 Jul; 167(1): 165–173. PubMed Abstract \| Publisher Full Text\n\nDanila MI, Hughes LB, Brown EE, et al.: Measurement of erythrocyte methotrexate polyglutamate levels: ready for clinical use in rheumatoid arthritis?. Curr Rheumatol Rep. 2010 Oct; 12(5): 342–347. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Rotte MC , den Boer E , de Jong PH , et al.: Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in patients with rheumatoid arthritis. Ann Rheum Dis. 2015 Feb; 74(2): 408–414. PubMed Abstract \| Publisher Full Text\n\nStamp LK, O'Donnell JL, Chapman PT, et al.: Methotrexate polyglutamate concentrations are not associated with disease control in rheumatoid arthritis patients receiving long-term methotrexate therapy. Arthritis Rheum. 2010 Febs; 62(2): 359–368. PubMed Abstract \| Publisher Full Text\n\nHobl EL, Jilma B, Erlacher L, et al.: A short-chain methotrexate polyglutamate as outcome parameter in rheumatoid arthritis patients receiving methotrexate. Clin Exp Rheumatol. 2012 Mar-Apr; 30(2): 156–163. PubMed Abstract\n\nGuo Q, Wang Y, Xu D, et al.: Rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies. Bone Res. 2018; 6: 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKushner I: C-reactive protein in rheumatology. Arthritis Rheum. 1991 Aug; 34(8): 1065–1068. PubMed Abstract\n\nAletaha D, Neogi T, Silman AJ, et al.: 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 2010 Sep; 62(9): 2569–2581. PubMed Abstract \| Publisher Full Text\n\nWells G, Becker JC, Teng J, et al.: Validation of the 28-joint Disease Activity Score (DAS28) and European League Against Rheumatism response criteria based on C-reactive protein against disease progression in patients with rheumatoid arthritis, and comparison with the DAS28 based on erythrocyte sedimentation rate. Ann Rheum Dis. 2009 Jun; 68(6): 954–960. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAletaha D, Ward MM, Machold KP, et al.: Remission and active disease in rheumatoid arthritis: defining criteria for disease activity states. Arthritis Rheum. 2005 Sep; 52(9): 2625–2636. PubMed Abstract \| Publisher Full Text\n\nTakahashi C, Kaneko Y, Okano Y, et al.: Association of erythrocyte methotrexate-polyglutamate levels with the efficacy and hepatotoxicity of methotrexate in patients with rheumatoid arthritis: a 76-week prospective study. RMD Open. 2017; 3(1): e000363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaylor PC, Balsa Criado A, Mongey AB, et al.: How to Get the Most from Methotrexate (MTX) Treatment for Your Rheumatoid Arthritis Patient?-MTX in the Treat-to-Target Strategy. J Clin Med. 2019 Apr 15; 8(4). Epub 20190415. PubMed Abstract \| Free Full Text Publisher Full Text \|\n\nSmolen JS, Aletaha D, Bijlsma JW, et al.: Treating rheumatoid arthritis to target: recommendations of an international task force. Ann Rheum Dis. 2010 Apr; 69(4): 631–637. PubMed Abstract \| Free Full Text Publisher Full Text \|\n\nRaimondi MV, Randazzo O, La Franca M, et al.: DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents. Molecules 2019 Mar 22; 24(6). Epub 20190322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriedman B, Cronstein B: Methotrexate mechanism in treatment of rheumatoid arthritis. Joint Bone Spine 2019 May; 86(3): 301–307. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan ES, Cronstein BN: Mechanisms of action of methotrexate. Bull Hosp Jt Dis (2013) 2013; 71 Suppl 1: S5–S8. PubMed Abstract\n\nYang JY, Webster-Clark M, Lund JL, et al.: Propensity score methods to control for confounding in observational cohort studies: a statistical primer and application to endoscopy research. Gastrointest Endosc. 2019 Sep; 90(3): 360–369. PubMed Abstract \| Free Full Text\n\nden Boer E , de Rotte MC , Pluijm SM, et al.: Determinants of erythrocyte methotrexate polyglutamate levels in rheumatoid arthritis. J Rheumatol. 2014 Nov; 41(11): 2167–2178. PubMed Abstract \| Publisher Full Text" }	[ { "id": "123625", "date": "21 Feb 2022", "name": "Slobodan M Janković", "expertise": [ "Reviewer Expertise Clinical pharmacology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors made an observational study trying to correlate MTX PG levels with disease activity of RA (as measured by a clinical score). The topic is of general interest, and the study brings results with practical significance. The manuscript is well written, and merits acceptance for publication. However, there are a few issues that should be corrected:\nIn the Methods section the authors should state precisely how they measures the MTX PG levels in erythrocytes. As it is written now, it is not clear whether the MTX PG levels were measured in erythrocytes or in full blood.\n\nNumber of patients is small, so it is critical that statistical methods were used properly. The authors should state whether assumptions of multivariate logistic regression were met. Also, what was the categorical outcome used as dependent variable of the regression? Finally, quality of the regression model should be stated (Hosmer Lemeshow test, Cox and Snellen...).\n\nSomething should be said about adherence of the patients to the therapy. Was there any method used to check for adherence? If not, mention this in the Limitation paragraph.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "123627", "date": "23 Feb 2022", "name": "Andri Frediansyah", "expertise": [ "Reviewer Expertise Pharmaco Biology/Analytical Chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe researchers looked at 34 people with rheumatoid arthritis (RA) to see if there was a link between MTX-PG levels and how active their RA was. There were two women and 32 men in the study. The subject matter is of general interest, and the study yields useful information. There are, however, a few issues that should be addressed:\n1) Please specify the date, duration, and months of the experiment.\n2) Please verify the following statement: \"low disease activity, <3.2–5.1\". Is this correct?\n3)The methods section is unclear. Please describe it in detail. Is there a particular type of blood (whole blood, red, or white blood cells) that you used in the study? Additionally, please provide detailed information about the centrifugation parameters, such as time, temperature, and g-force/RCF (g). Prior to analysis, is the blood subjected to any special treatment?\n4) Please rewrite the section on chromatography measurement and analysis in detail. Include the HPLC specification and brand; column details (including particle size, pore size, inner diameter, and length); ammonium hydrochloride concentration and pH; solvent B composition (or A, if any); and the reference you cited.\n5) Did you combine ammonium bicarbonate and ammonium chloride, and if so, in what proportion? Which detector (UV/CAD/MS) did you use? If UV/DAD, at what wavelength did you adjust the detector?\n6) Please specify the brand of the MTX-PG3 standard and the R2 (nmol) value of the standard you used.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "123624", "date": "02 Mar 2022", "name": "Talha Bin Emran", "expertise": [ "Reviewer Expertise Public health", "Immunology", "Natural Product Chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle: Correlation between serum methotrexate-polyglutamate 3 (MTX-PG3) level and disease activity in rheumatoid arthritis patients: A prospective cohort study\nMinor comments: Although the article has scientific rigor, several minor flows need to be improved before publication:\n1. The abstract section is unsuitable—no focus point in the abstract section.\n2. \"Nevertheless, a prospective study with a higher number of patients is needed to confirm this finding.\" Is this necessary?\n3. Authors are suggested to use the full form when used for the first time throughout the manuscript.\n4. The aim of the study should be written as the last paragraph of the introduction.\n7. MTX treatment and follow-up: How was this selected?\n8. Receiver Operating Characteristics (ROC) analysis: Please describe in further detail.\n9. \"Further analysis using the ROC curve showed that MTX-PG3 level…\" needs more insights with relevant references.\n10. Presentation of figures is good.\n11. Figure legends are appropriate and self-explanatory.\n12. The conclusion needs to address future perspectives.\n13. Spacing, punctuation marks, grammar, and spelling errors should be reviewed thoroughly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-187
https://f1000research.com/articles/10-1133/v1	09 Nov 21	{ "type": "Research Article", "title": "Data analytics competency and religiosity influence on external auditors’ performance in Malaysia", "authors": [ "Nahariah Jaffar", "Abdul Aziz Bin Ahmad", "Noor Adwa Sulaiman", "Abdul Aziz Bin Ahmad", "Noor Adwa Sulaiman" ], "abstract": "Background - Data analytics can support the external auditors’ judgements. However, little is known about the external auditors’ data analytics competency. Likewise, role of religiosity in enhancing the external auditors’ performance is also inadequately investigated. This study examined: 1) the effects of data analytics competency on the external auditors’ performance, and 2) the moderating effects of religiosity on data analytics competency and external auditors’ performance relationship. Methods – Survey was conducted on 201 external auditors. Data analytics competency dimensions, namely, personal capabilities, professional expertise, technical skills, technologies and tools expertise were examined. Religiosity was measured by level and dimension (faith, virtue and optional). Results – Data analytics competency (personal capabilities) has a positive significant effect on the Muslim external auditors’ performance. However, data analytics competency does not affect the performance of non-Muslim external auditors. Level of religiosity has significant moderating effect on the relationship between data analytics competency (technologies and tools expertise) and Muslim external auditors’ performance. Nonetheless, level of religiosity does not moderate the relationship between data analytics competency and the performance of non-Muslim external auditors. Religiosity (virtue) has significant moderating effect on the relationship between data analytics competency (personal capabilities) and Muslim external auditors’ performance. Meanwhile, religiosity (faith) has significant moderating effect on the relationship between data analytics competency (technologies and tools expertise) and non-Muslim external auditors’ performance. Conclusion – This study demonstrates that data analytics competency and religiosity can influence the external auditors’ performance.", "keywords": [ "Data analytics", "competency", "religiosity", "external auditors", "performance", "Malaysia", "Muslim", "non-Muslim" ], "content": "Introduction\n\nIn this Fourth Industrial Revolution era, external auditors (EAs) are expected to acquire data analytics competency (DAC) because it can strengthen audit quality (e.g. ICAEW, 2016; Goh, 2017). However, evidence regarding the DAC of EAs and its impact on the external auditors’ performance (EAP) is limited (Yeo & Carter, 2017). Furthermore, literature suggests religiosity influences individual’s behaviour which includes commitment and ethics (e.g. Van Buren, Syed & Mir, 2020). Nonetheless, little is known about the role of religiosity in enhancing the EAP in the context of DAC. Thus, this study aims: (1) to investigate the influence of DAC on the EAP, and (2) to examine the moderating effect of religiosity on the relationship between DAC and the EAP.\n\n\nCompetence performance theory and hypotheses\n\nThis study adopts Competence Performance Theory (CPT) by Korossy (1999). CPT establishes a connection between competency and performance level. In this study, perceived DAC (personal capabilities, professional expertise, technical skills, and technologies and tools expertise) aids to predict the EAP. Low in certain DAC would affect EAP. Thus, this study hypothesises:\n\nDAC (personal capabilities) has positive effects on the Muslim/non-Muslim-EAP.\n\nDAC (professional expertise) has positive effects on the Muslim/non-Muslim-EAP.\n\nDAC (technical skills) has positive effects on the Muslim/non-Muslim-EAP.\n\nDAC (technologies and tools expertise) has positive effects on the Muslim/non-Muslim-EAP.\n\nAli, Baluch and Mohamed Udin (2015) proposed a moderating role of religiosity on the relationship between technology readiness and diffusion of electronic commerce. This suggests that religiosity may influence the consistency of behaviour exhibited by the EAs. It is predicted that high DAC and level of religiosity will enhance the EAP. Therefore, this study further hypothesizes:\n\nReligiosity (level/dimension) moderates the relationship between DAC (personal capabilities) and the Muslim/non-Muslim-EAP.\n\nReligiosity (level/dimension) moderates the relationship between DAC (professional expertise) and the Muslim/non-Muslim-EAP.\n\nReligiosity (level/dimension) moderates the relationship between DAC (technical skills) and the Muslim/non-Muslim-EAP.\n\nReligiosity (level/dimension) moderates the relationship between DAC (technologies and tools expertise) and the Muslim/non-Muslim-EAP.\n\n\nMethods\n\nSurvey questionnaires (Underlying data) (Jaffar et al., 2021) were distributed among final year accounting students at one private university in Malaysia. There were 201 students and all of them participated in the survey. These students had undergone a six-month auditing internship program thus deemed suitable to be used as proxies for EAs. This approach is similar with Ashton and Kramer (1980) who also used students as surrogates for auditors.\n\nDAC was measured by analysing: personal capabilities, professional expertise, technical skills, and technologies and tools expertise as proposed by Strengell’s (2017) (Underlying data) (Jaffar et al., 2021). Religiosity (beliefs and practices) was adapted from Mahdzan, Zainudin, Che Hashim and Sulaiman’s (2017). EAP was measured using Asare and Cianci’s (2009) technique, where respondents were given a hypothetical inventory audit case and asked to indicate the likelihood of recommending the inventory to be written off. A pilot test with 50 students revealed that the questionnaire needed no changes. Finally, the questionnaires were sent to the actual respondents.\n\nModels of this study were as follows:\n\nModel 1:\n\nModel 2:\n\nModel 3:\n\nModel 1 depicts the relationship between DAC and EAP. Level of religiosity (Model 2) and religiosity dimensions (Model 3) were included to test the moderating effects of religiosity on the relationship between DAC and EAP.\n\n\nResults and statistical analysis\n\nThis study used SPSS Version 26 to analyse the data. Specifically, linear regression was applied to test all the three models of the study. The linear regression was performed separately for Muslim and non-Muslim EAs. Exploratory factor analysis (EFA) was performed to identify the latent constructs of religiosity, and means analysis was used to determine the level of religiosity. Details of the data analysis were presented in the following sections.\n\nLinear regression was performed separately for Muslim and non-Muslim EAs. For Muslim EAs, the result exhibited a close to moderate level of R square (42.2%) which signified that variation of the Muslim-EAP was almost moderately explained by their DAC. DAC statistically (p=0.000) predicted the Muslim-EAP. Coefficient results showed that DAC (personal capabilities) was the main predictor (p = 0.000) of the Muslim-EAP. Results of Model 1 for non-Muslim EAs revealed a weak level of R square (1.9%) denoting that the variation of the non-Muslim-EAP was weakly explained by their DAC. ANOVA displayed p = 0.536 indicating that DAC did not statistically predict the non-Muslim-EAP.\n\nUsing factor loading of 0.6, EFA was performed to identify the latent constructs of religiosity (Hair, Sarstedt, Hopkins & Kuppelwieser, 2014). Only 21 items were used to represent the latent constructs of religiosity for Muslim EAs. The names of the factors were the same as those used by Mahdzan et al. (2017) who adopted Wan Ahmad, Ab. Rahman, Ali and Che Seman’s (2008) and Tiliouine and Belgoumidi’s (2009) measurement scale. Factor one, two and three demonstrated high reliability scores with Cronbach’s alpha between 0.843 and 0.971. This study used 19 items to capture the non-Muslim EAs’ religiosity constructs. The same procedures were applied to the religiosity data of the non-Muslim EAs. Based on the EFA results, five items were discarded because of low factor loadings. The remaining 14 items produced a five-factor solution. Following the test, factor one produced a Cronbach’s alpha value of above 0.8 which was considered good. The Cronbach’s alpha values for factors two and three were 0.752 and 0.629, respectively, which were considered acceptable. Finally, only 12 items were used to represent the latent constructs of religiosity for the non-Muslim EAs.\n\nMuslim religiosity level was measured by the religiosity strength represented by Casual, Moderate and Devout. Similar to Mahdzan et al. (2017), a grand mean was computed as follows:\n\n• Casual - mean scores (μ) were 0.5; standard deviation of 2.915 (μ ≤ 2.915)\n\n• Moderate - mean scores were between 2.925 and 3.415 (2.915 < μ > 3.415)\n\n• Devout - mean scores above 3.415 (≥ 3.415)\n\nUsing similar procedures, the non-Muslim EAs’ religiosity strength was computed as follows:\n\n• Casual - mean scores were 0.5; standard deviation of 2.411 (μ ≤ 2.411)\n\n• Moderate - mean scores were between 2.411 and 2.911 (2.411 < μ > 2.911)\n\n• Devout - mean scores above 2.911 (≥ 2.911)\n\nIn Model 2, linear regression on DAC, level of religiosity and Muslim-EAP exhibited moderate R square (65%) signifying that variation of the Muslim-EAP was moderately explained by DAC and level of religiosity. The ANOVA results displayed p = 0.000 indicating that the Muslim-EAP was significantly predicted. The subsequent results showed that level of religiosity has significant moderating effect (p = 0.054) on the relationship between DAC (technologies and tools expertise) and the Muslim-EAP. The technologies and tools expertise, however, has statistically significant negative main effect on the Muslim-EAP at p = 0.030. This result denoted that the higher the DAC (technologies and tools expertise), the lower the Muslim-EAP. The inclusion of the level of religiosity has improved the overall model fit as evidenced by the higher R square (65%) compared to Model 1’s R square (49%).\n\nRegression results for DAC, level of religiosity and non-Muslim-EAP indicated low R square (4.2%) demonstrating that variation of the non-Muslim-EAP was weakly explained by the DAC and level of religiosity. ANOVA results showed p = 0.556 indicating that the non-Muslim-EAP was not statistically predicted. The inclusion of the level of religiosity slightly improved the overall model fit as evidenced by the higher R square (4.2%) compared to Model 1’s R square (1.9%). Nonetheless, the R square is still considered as low.\n\nWan Ahmad et al. (2008) introduced four dimensions of Islamic religiosity: Faith, Virtue, Obligation, and Optional. Moderating effects of religiosity dimensions on the relationship between DAC and the EAP were predicted in Model 3 using linear regression analysis.\n\nRegression results showed only the Virtue dimension has significant effect on the relationship between DAC and the Muslim-EAP but not Faith and Optional dimensions. R square = 61.9% suggested that variation of the Muslim-EAP was moderately explained by DAC and Virtue, as supported by ANOVA results of p = 0.001. Subsequent results showed that DAC (personal capabilities) has statistically significant positive main effects on Muslim-EAP at p = 0.011, while DAC (technologies and tools expertise) has statistically significant negative main effects on the Muslim-EAP at p = 0.10 (p < 0.1). The result also demonstrated that Virtue as a dimension of religiosity has significant moderating effect (p = 0.098, using significant level of p < 0.1) on the relationship between DAC (personal capabilities) and the Muslim-EAP. However, the coefficient is -2.084 which denoted the negative direction of the moderating effect of Virtue. In other words, the higher the religiosity (Virtue) the more negative the effect of DAC (personal capabilities) on the Muslim-EAP. Nonetheless, it can be concluded that Virtue as a dimension of religiosity has improved the overall model fit as evidenced by the higher R square (61.9%) compared to Model 1’s R square (49%).\n\nFor non-Muslim EAs, the regression results showed only Faith dimension has significant effect on the relationship between DAC and the non-Muslim-EAP but not Virtue and Optional dimensions. The regression result for Faith showed a moderate R square (8.5%) suggesting that variation of the non-Muslim-EAP was weakly explained by DAC and Faith as a dimension of religiosity. The ANOVA results displayed p = 0.076 (p < 0.1), signifying that the non-Muslim-EAP was significantly predicted. The subsequent results showed that DAC (technologies and tools expertise) has statistically significant positive main effects on the non-Muslim-EAP at p = 0.007. Faith as a dimension of religiosity has a significant moderating effect (p = 0.002) on the relationship between DAC (technologies and tools expertise) and the non-Muslim-EAP. However, the coefficient is −0.624 which denoted the negative direction of the moderating impact of Faith. In other words, the higher the religiosity (Faith) the more negative the effect of DAC (technologies and tools expertise) on the non-Muslim-EAP. Nevertheless, the inclusion of Faith as a dimension of religiosity has improved the overall model fit as shown by the higher R square (8.5%) compared to Model 1’s R square (1.9%). Nonetheless, the R square is still considered as very low.\n\n\nDiscussion\n\nIn summary, H1a is accepted for Muslim EAs but not for the non-Muslim EAs. This finding indicates that DAC (personal capabilities) improves the Muslim-EAP. This suggests that personal qualities such as creativity and stress tolerance can improve Muslim EAs performance. This finding corroborates with CPT and Ebrahimi and Hassanein (2018) who demonstrated that DAC has a significant positive relationship with firm’s decision-making performance. As for non-Muslim EAs, H1d is accepted (under Model 3). This finding demonstrates that DAC (technologies and tools expertise) enhances the non-Muslim EAs performance. Obviously, expertise in data analytics technology and methods can enhance the performance of the non-Muslim EAs. This finding is consistent with CPT and Ghasemaghaei, Ebrahimi and Hassanein (2018) regarding the potential impact of DAC on performance. Although the finding for H1d for the Muslim EAs is significant, the relationship is negative (under Model 2). The finding suggests that the higher the skill in data analytics technologies and tools, the lower the performance of the Muslim EAs. This could be due to the nature of EAs' work which relies generally on professional opinion. In addition, Omitogun and Al-Adeem (2019) found that EAs lack relevant technical skills and were unfamiliar with related data analysis tools, except Excel. Both H1b and H1c are not accepted. These findings demonstrate that DAC (professional expertise and technical skills) does not influence the EAP. Professional expertise requirements such as problem-solving skills and reporting are among expertise already required by the profession. This finding corroborates Weber’s (2020) revelation that the EAs were not well prepared for data analytics and that they only had a moderate sense of urgency to become competent in analytics, owing to their employers’ lack of demand for analytics.\n\nH2a, H3a, and H4a are not accepted, implying that level of religiosity has no influence on the relationship between DAC (personal capabilities, professional expertise, and technical skills) and the EAP which is inconsistent with prior research by Ali et al. (2015). The insignificant results may be due to the fact that in the audit profession, the EAs are required to comply with the Professional Code of Ethics. For Muslim EAs, H5a is accepted, but not for non-Muslim EAs. This suggests that the relationship between DAC (technologies and tools expertise) and the Muslim-EAP is moderated by level of religiosity. This finding also shows that the more religious the Muslim EAs are – which indicates that they are honest, ethical or trustworthy – the greater the favourable influence of DAC (technologies and tools expertise) on the Muslim-EAP.\n\nH3b and H4b are not accepted, denoting that religiosity (Faith, Virtue and Optional) does not moderate the relationship between DAC (professional expertise and technical skills) and the EAP. These findings do not corroborate Ali et al. (2015) and this may be due to the fact that the EAs already conform to the Code of Professional Ethics. In the case of H2b, however, religiosity (Virtue) significantly moderated the relationship between DAC (personal capabilities) and the Muslim-EAP, but not for non-Muslim EAs. Nonetheless, the direction is not consistent with the prediction of this study that the more religious Muslim EAs are, the lesser the impact of DAC (personal capabilities) on their performance. Likewise, for H5b, religiosity (Faith) significantly moderated the relationship between DAC (technologies and tools expertise) and the non-Muslim-EAP but not for Muslim-EAP. Nevertheless, the direction is not consistent with the prediction of this study that the more religious non-Muslim EAs are, the lesser the impact of DAC (technologies and tools expertise) on their performance.\n\nThis study acknowledged one limitation of this study that was the use of students as proxy for external auditors which could cause problems with generalizability. Nevertheless, because these students had undergone a six-month internship at the accounting firms, this constraint was minimised. As a result of this exposure, they were qualified to act as external auditors' surrogates.\n\n\nConclusion\n\nIn conclusion, this study reveals that having DAC is critical for EAs because it can improve their performance. This requires the EAs to acquire data analytics skills for rendering audit services. Furthermore, accounting firms should make data analytics training a priority for their staff. Additionally, religiosity, which has been inadequately tested in DAC literature, provides insights into the significant role of religiosity in EAP. Future research could investigate the cost and benefits of using data analytics in enhancing audit efficiency.\n\n\nEthical approval\n\nThis research received an ethical approval from the Research Ethics Committee (REC) of Multimedia University (Approval Number EA2302021).\n\n\nData availability\n\nFigshare: Data Analytics Competency and Religiosity Influence on External Auditors’ Performance in Malaysia\n\nhttps://doi.org/10.6084/m9.figshare.16803472 (Jaffar et al. 2021)\n\nThis project contains the following underlying data:\n\nData (1): The dataset comprises of demographic data of the external auditors, data analytics competency items, religiosity items and performance.\n\nData file (2): Questionnaire used to collect data on the external auditors’ demographic profile, data analytics competency, religiosity and performance.\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\nNahariah Jaffar, Abdul Aziz Ahmad and Noor Adwa Sulaiman were involved in conceptual framework, methodology, analysis, discussion, and writing of the article.", "appendix": "References\n\nAli B, Baluch N, Mohamed Udin Z: The moderating effect of religiosity on the relationship between technology readiness and diffusion of electronic commerce. Mod. Appl. Sci. 2015; 9(12): 52–60. Publisher Full Text\n\nAsare KS, Cianci MA: The effect of goals on auditors' judgments and their perceptions of and conformity to other auditors' judgments. Manag. Audit. J. 2009; 24(8): 724–742. Publisher Full Text\n\nAshton RH, Kramer SS: Students as surrogates in behavioral research: some evidence. J. Account. Res. 1980; 18(1): 269–177. Publisher Full Text\n\nGhasemaghaei M, Ebrahimi S, Hassanein K: Data analytics competency for improving firm decision making performance. J. Strateg. Inf. Syst. 2018; 27(1): 101–113. Publisher Full Text\n\nGoh C: Are you ready? Data analytics is reshaping the work of accountants. Cfo. Research Collection School of Accountancy.2017.\n\nHair JF Jr, Sarstedt M, Hopkins L, et al.: Islamic religiosity and portfolio allocation: The Malaysian context. Int. J. Islam. Middle East. Financ. Manag. 2014; 10(3): 434–452. In Mahdzan, S.N., Zainudin, R., Che Hashim, R. & Sulaiman, N. (2017).\n\nICAEW: Data analytics for external auditors. International Auditing Perspectives: An International Accounting, Auditing & Ethics initiative.2016.\n\nJaffar N, Ahmad AA, Sulaiman NA: F1000R~Data.XLS. figshare. Dataset. 2021. Publisher Full Text\n\nKorossy K: Modelling knowledge as competence and performance. Psychology Press; 1999.\n\nMahdzan SN, Zainudin R, Che Hashim R, et al.: Islamic religiosity and portfolio allocation: The Malaysian context. Int. J. Islam. Middle East. Financ. Manag. 2017; 10(3): 434–452. Publisher Full Text\n\nOmitogun A, Al-Adeem K: Auditors’ perceptions of and competencies in Big Data and data analytics: An empirical investigation. International Journal of Computer Auditing. 2019; 1(1): 92–113.\n\nStrengell T: Competitiveness from data and analytics: Required competency in organization. University of Jyväskylä; 2017. [Unpublished PhD Thesis].\n\nTiliouine H, Belgoumidi A: An exploratory study of religiosity, meaning in life and subjective wellbeing in Muslim students from Algeria. Appl. Res. Qual. Life. 2009; 4(1): 109–127. Publisher Full Text\n\nVan Buren JH III, Syed J, Mir R: Religion as a Macro Social Force Affecting Business: Concepts, Questions, and Future Research, Business & Society.2020; 59(5): 799–822.\n\nWan Ahmad WM, Ab. Rahman A, Ali NA, et al.: Religiosity and banking selection criteria among Malays in Lembah Klang. Shariah J. 2008; 16(2): 279–304.\n\nWeber J: CPAs as data analysts: How Louisiana accountants respond to data-driven business paradigms. Liberty University, School of Business; 2020. [Unpublished Doctor of Business Administration].\n\nYeo AC, Carter S: Segregate the wheat from the chaff enabler: Will Big Data and data analytics enhance the perceived competencies of accountants/auditors in Malaysia?. J. Self-Gov. Manag. Econ. 2017; 5(3): 28–51. Publisher Full Text" }	[ { "id": "99722", "date": "13 Dec 2021", "name": "Prem Lal Joshi", "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 issues studied in this research are contemporary in auditing research, and limited work has been undertaken so far in this area. Therefore, any research on the effects of data analytics competency on the external auditors’ performance is very welcome. The theme and research questions investigated in this study are highly relevant. Additionally, it is interesting to know the moderating effects of religiosity on data analytics competency and external auditors’ performance relationship.\nThis research uses a survey method which is acceptable. The sample size is quite large. The data analytics competency dimensions are indicated by personal capabilities, professional expertise, technical skills, technologies and tools expertise, which may be considered as relevant measures. The religiosity variable is measured by level and dimension (faith, virtue and optional). The theory applied in this research is Competence Performance Theory (CPT) as laid out by Korossy (1999), which is also relevant to this type of research.1 The hypotheses formulated are duly supported by prior research.\nThe study findings state that data analytics competency (personal capabilities) has a positive significant effect on the Muslim external auditors’ performance. However, data analytics competency does not affect the performance of non-Muslim external auditors. Level of religiosity has a significant moderating effect on the relationship between data analytics competency (technologies and tools expertise) and Muslim external auditors’ performance. However, the level of religiosity has no moderating effect on the relationship between data analytics competency and the performance of non-Muslim external auditors.\nMy main concern is that the authors have not provided clear reasons or justifications for these differences in findings between Muslim external auditors and non-Muslim external auditors. Do both the groups acquire different education training and skills with respect to DAC in Malaysia? Why does this difference occur? Can these findings be generalized to other environments?\nI recommend the manuscript for approval subject to addressing the above minor 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": [ { "c_id": "7803", "date": "14 Feb 2022", "name": "nahariah jaffar", "role": "Author Response", "response": "Dear Prof Thank you for your time in reviewing our manuscript. We are greatly appreciative of the insightful comments that you have provided. Our responses to your comments are as follows: 1- Justification for the differences in findings between Muslim external auditors and non-Muslim external auditors. Our response: The revised version provided the justification (in the discussion section) that one possible reason might be due to the level of support provided by the auditing firms. Hence, further research is needed to understand other possible factors affecting the DAC and subsequently the EAP. 2- Generalizability of the findings to other environments. Our responses: In the discussion section of the revised version, it is stated that the study provided an initial investigation into the effects of religiosity on EAP. Future research might want to incorporate other contributing factors affecting EAP such as organizational culture to understand the effects of various factors affecting performance in other environment or setting. We hope that our responses are acceptable and look forward to your feedback. Thank you again for your time in reviewing this manuscript." } ] }, { "id": "99723", "date": "18 Jan 2022", "name": "Khalid Al-Adeem", "expertise": [ "Reviewer Expertise Development of Accounting Thought", "IFRS", "Accounting Research Methodology", "Epistemology", "Auditor Independence" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe hypotheses are not deduced from a theory or at least literature. For the hypothetico-deduction to be properly implemented, hypotheses need to be deduced from a theory/theories or at least prior literature on the phenomenon being investigated. Without doing so, the work is merely speculative\n\nFurthermore, the linear regression does not count for the error term in models. However, I see that the authors included the error term in the three models. The author could have employed Structural Equation Modeling (SEM) to count for the error term, and test the three models once as opposed to test them separately.\n\nWith regard to identifying the “latent constructs of religiosity”, I suggest further performing Confirmatory Factor Analysis (CFA) to confirm the loading and assure the existence of such a contract. That is, to validate what the Exploratory Factor Analysis (EFA) suggests.\n\nThere are grammatical errors in this paper, it should be gone over again and these should be corrected.\n\nThe authors write, 'Meanwhile, religiosity (faith) has significant moderating effect on the relationship between data analytics competency (technologies and tools expertise) and non-Muslim external auditors’ performance',\nI suggest writing the statement this way:\n\n“Meanwhile, religiosity (faith) has a significant moderating effect on the relationship between data analytics competency (technologies and tools expertise) and non-Muslim external auditors’ performance”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7804", "date": "14 Feb 2022", "name": "nahariah jaffar", "role": "Author Response", "response": "Dear Prof Thank you for reviewing our manuscript. We are grateful for the insightful comments you have shared. The following are our responses to your comments: 1- Hypotheses not deduced from theory Our response: A revision was made to improve the clarity of the hypotheses development section. This study adopted Competence Performance Theory (CPT) by Korossy (1999) to predict the effects of DAC on the EAP. In addition, additional literature were added to explain the moderating role of religiosity. 2- Adoption of SEM Our response: The revised manuscript retained the existing data analysis because the current software used in the study was able to analyse the current data and achieved the objectives of the study. 3- Grammatical error Our response: the manuscript has been sent for English proof reading. 4- To improve one statement in the abstract. Our response: We have made the necessary changes. We hope that these responses are acceptable and look forward to your feedback. Thank you again for your time in reviewing our manuscript." } ] }, { "id": "99721", "date": "24 Jan 2022", "name": "Amal Hayati Ahmad Khair", "expertise": [ "Reviewer Expertise My primary research interests are accounting fraud/bribery/corruption", "fraudulent accounting practices", "auditing", "ethics", "and 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\nMy comments:\nThis is a potentially interesting paper. The author/s have an interesting context and study involving data analytics competency and religiosity, in which the former is developed based on four dimensions of competency (i.e. personal capabilities, professional expertise, technical skills, and technology and tools expertise) and the latter, as the moderating factor, is developed based on two measurements of religiosity (i.e. level and dimension), in which their impact were tested on external auditors’ performance in Malaysia.\nIt addresses an important topic, especially since some have proposed that religiosity positively influences individual performance1 and organisational behaviour and thus has substantial economic implications.2 Similarly, some have argued that competency has significant impact on performance.3 However, as currently written, the paper seems to be at a very early stage of development and thus considerable development is needed. Despite the rather small temporal and spatial extent of the study on external auditors’ performance (EAP), it should make an important contribution of behavioural studies especially in the auditing within Islamic context.\nThe paper has initiated discussions on the issues associated with competency and religiosity on the EAP. However, it exhibits a lack of organisation and coherent arguments in some instances. For example, starting from page 3 onwards, some of the ideas and arguments have not been developed adequately, especially with the hypotheses’ development. Although the paper has attempted to develop the hypotheses with reference to the literature, the arguments were inadequately discussed and have not been linked properly in some instances. For example, under the ‘Dimensions of Religiosity’ section on page 5, four dimensions have been used for Islamic religiosity (i.e. faith, virtue, obligation, optional), but these clash with the abstract, which included only three dimensions (i.e. faith, virtue and optional). Another example, the two measurements of religiosity (i.e. level and dimension), which were introduced in ‘Competence performance theory and hypotheses’ section on page 3, could have been explained clearly how these two dimensions were utilised as moderating factors in the study. Some parts of the methodology sections could have been explained better, for example what it is meant by casual, moderate and devout (refer page 4) for religiosity strength and its link to the EAP.\nIn term of argument and methodology, the paper is built on appropriate known concepts, and research is properly designed with correct methodology. However, the discussion on research design and sample could have explained the distribution between Muslim and non-Muslim external auditors. The results could have been presented in tables so that they could be understood and communicated better. Nevertheless, the results have been analysed appropriately and conclusions have been adequately tied together the other elements of the paper in term of the research objective, literature reviews and results.\nIn general, the paper clearly expresses its case by taking into considerations all aspects in term of expected knowledge of readers and appropriate language used. However, it contains a number of typographical errors, occasional stylistic or grammatical flaws, corrections to references, etc. Further proof reading would benefit the audience.\nComments to the Author/s:\nThe subject of your paper is important and the empirical material you have collected holds potential. However, I believe the paper needs considerable development, particularly around the clarity of the concepts mobilised and model development and cannot be given the status approved until these revisions are made. The highlighted comments offer a number of important observations and suggestions for further improvement.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "7805", "date": "14 Feb 2022", "name": "nahariah jaffar", "role": "Author Response", "response": "Dear Prof. Thank you for reviewing our manuscript. We are grateful for the insightful comments you have shared. The following are our responses to your comments: 1- Lack of organisation and coherent arguments in hypotheses development. Our response: A revision was made to improve the clarity of the hypotheses development section. Additional literature was added to explain the moderating role of religiosity. 2- Unclear on Dimensions of Religiosity. Clearer explanation was made in the religiosity section regarding the adoption of religiosity dimensions in the study that was based on high factor loading. 3- Unclear on Level of Religiosity. Clearer explanation was provided in the Level of Religiosity section regarding the meaning of the three levels of religiosity (casual, moderate, devout). We hope that these responses are acceptable and look forward to your feedback. Thank you again for your time in reviewing our manuscript." } ] } ]	1	https://f1000research.com/articles/10-1133
https://f1000research.com/articles/10-22/v1	14 Jan 21	{ "type": "Brief Report", "title": "Runcer-Necromancer: a method to rescue data from an interrupted run on MGISEQ-2000", "authors": [ "Anna Pavlova", "Vera Belova", "Robert Afasizhev", "Irina Bulusheva", "Denis Rebrikov", "Dmitriy Korostin", "Robert Afasizhev", "Irina Bulusheva", "Denis Rebrikov", "Dmitriy Korostin" ], "abstract": "During the sequencing process, problems can occur with any device, including the MGISEQ-2000 (DNBSEQ-G400) platform. We encountered a power outage that resulted in a temporary shutdown of a sequencer in the middle of the run. Since barcode reading in MGISEQ-2000 takes place at the end of the run, it was impossible to use non-demultiplexed raw data. We decided to completely use up the same cartridge with reagents and flow cell loaded with DNB and started a new run in a shortened custom mode. We figured out how the MGISEQ-2000 converts preliminary data in .cal format into .fastq files and wrote a script named “Runcer-Necromacer” for merging .fastq files based on the analysis of their headers (available online: https://github.com/genomecenter/runcer-necromancer). Read merging proved to be possible because the MGISEQ-2000 flow cell has a patterned structure and each DNB has invariable coordinates on it, regardless of its position on the flow cell stage. We demonstrated the correctness of data merging by comparing sample analysis results with previously obtained .fastq files for them. Thus, we confirmed that it is possible to restart the device and save both parts of the interrupted run.", "keywords": [ "MGISEQ-2000", "DNBSEQ-G400", "NGS", "Paired-end sequencing", "fastq merging" ], "content": "Introduction\n\nAt the end of 2017, Chinese company MGI Tech presented the MGISEQ-2000 sequencing platform1, promoting it as a device for large and medium scale genome sequencing. MGISEQ is specific in harnessing cPAS sequencing technology and using nanoballs (DNB) generated from circular molecules of DNA library by rolling circle replication2. MGISEQ is compatible with a wide range of reagents for sequencing in SE50, SE100, SE400, PE100, PE150, and PE200 modes. MGISEQ-2000 provides the quality of sequencing comparable with that of the Illumina platform3–6.\n\nThe first MGISEQ-2000 sequencer in Russia was installed in our lab (Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Medical University) in February 2019, and we run it once a week in the paired-end 150 mode (PE150). According to our experience, one PE150 run usually takes 68 hours if one flow cell is used at a time. During one of these runs, about 23.00 on Saturday, there was a failure of the Moscow power grid leading to a 50-minute blackout of a whole district including Pirogov Medical University. UPS battery storage was sufficient only for 20 extra minutes, then the sequencer turned off until the power was restored. Therefore, the device with loaded reagents remained in sleep mode for 35 hours until Monday. Before the instrument was switched off, it had performed 138 full cycles of forward read sequencing (run 27). The specific feature of the MGISEQ-2000 sequencing program is that it reads a barcode at the end of a run after it completes sequencing of forward and reverse reads. So, to a first approximation, the data obtained could not be demultiplexed as information on the barcodes was absent.\n\nAccording to the MGI Tech7 recommendations, after consulting with the MGI Tech service engineers, we were advised to dispose of the current tank with reagents as well as the flow cell and run the samples using new reagents. In the first place, it is linked to the high sensitivity of the MDA reagent to storage at +4°C as it loses its activity very quickly. We decided to continue the run using the reagents that had been loaded for the weekend and try to restore the data. Finally, we managed to rescue the data using the software from ZebraCall8 and our own script on C++, which is reported here https://github.com/genomecenter/runcer-necromancer.\n\n\nMethods\n\nWe prepared 3 pools of circularized libraries following the standard MGI Tech protocol9. Then we synthesized DNB, loaded a flow cell using the MGIDL-200H manual loader, prepared a sequencing cartridge from the MGISEQ-2000RS High-throughput Sequencing Set with User manual version: A2, and started sequencing on A-side in PE150 mode. Run 27 was aborted at the 139th cycle of the read-1 sequencing phase. After 35 h, we restarted the run (run 27_2) using the same sequencing cartridge and flow cell in a custom mode with the following parameters: read 1 for 12 cycles, read 2 for 151 cycles, Start phase: Sequencing (Figure 1). For summary reports generated by MGISEQ-2000 for lane 1 of runs 27 and 27_2, see Extended data: File S1, S2.\n\nThe generation of .fastq files containing forward reads for the interrupted run was performed using ZebraCall v28 framework (C\\:ZebraCallV2\\client.exe – the pathway to software on MGISEQ-2000), which transforms intermediate .cal files into fastq format and demultiplexes them using barcodes.\n\nThe appropriate work of ZebraCall requires a .txt file with barcode sequences used for demultiplexing. We created an empty file 'empty_barcode.txt' so that the last 10 nucleotides from 13 nucleotides that were read earlier would not be recognized as barcodes by ZebraCall.\n\nWe used the following command (we provide an example for lane 1):\n\n\n\nIt contains the options:\n\nthe access to the folder with .cal files\n\nrun_name — the name of a run\n\n139 – the number of completed sequencing cycles\n\n6 72 – the number of fields of view counted horizontally and vertically for a corresponding lane\n\n-B – a path to the file with barcodes\n\n-U – the number of a lane\n\n-F – fastq generation without generation of flow cell images\n\nAs a result, for each lane, we generated files 'run_name_L0N_read.fq.gz' where N is a lane number. Such file contained a read name and a sequence of 138 nucleotides long.\n\nMGISEQ-2000 employs a patterned flow cell, so each DNB in a cell has unique coordinates at X and Y axes which do not depend on flow cell localization in a device and are not changed if the flow cell is displaced. When the power of the sequencer was off, the vacuum pump was switched off as well. The coordinates of each read were saved in a header of a .fastq file (Figure 2). This allowed us to integrate the data on forward reads obtained before and after the instrument was off.\n\nAs a read number being used for forward and reverse reading is unique, we managed to combine the 138-nucleotide sequences obtained during the first run with the nucleotide sequences obtained during the second run based on the information on F.O.V Column, F.O.V Row, and read numbers. To achieve this, we created a C++ script, which can be accessed at GitHub https://github.com/genomecenter/runcer-necromancer. The instruction for script running can be found below and in the file README.md in the repository.\n\nThe script (http://doi.org/10.5281/zenodo.431680210) is executable on Linux (was tested on Ubuntu 20.04) with GCC compiler with C++17 support and zlib (apt-get install zlib1g-dev). First step is a building: you need to run build.sh script inside the root folder. SaveReads program recovers sample files by placing fixed files into the fixed directory inside current directory. It is important to check that there are no identical filenames between samples files. SaveReads accepts N+1 argument, where first argument is _undecoded.fq.gz (pool of non-demultiplexed reads) file from interrupted run, and next N arguments are standard samples files. Script SaveReads.py simplifies call to SaveReads. This file accepts pool of non-demultiplexed reads as its single argument. All files with _1.fq.gz ending from current folder will be taken as samples files.\n\n\nResults\n\nThe most important parameter for sequencing quality is the ratio of the data with the quality level of no less than Q30. The Q30 value and other quality metrics did not decrease dramatically in spite of a 35-hour stand by (Figure 3, Table 1).\n\nQ30 histograms for runs 27 (А) and 27_2 (B). The X-axis represents the number of sequencing cycles, the Y-axis represents the ratio of the data with the quality no less than Q30 (%). Blue arrows in histogram B indicate the cycles which reverse reading and barcode reading started.\n\nThis implies that storing a loaded cartridge for 35 h leads to its decline, however, it can be still used for sequencing.\n\nTo check if merging reads from different runs was correct, we compared 7 samples of whole-exome sequencing from runs 27 and 27_2 with the data from the same samples obtained in run 22. We used the distribution of the size of an insert between left and right reads and the ratio of reads having the insert size exceeding 1000 nucleotides as control metrics. If read merging had been performed with errors, the portion of the reads mapped to various genome regions would have significantly increased. See Figure 4 for the distribution of an insert size for sample LWX777 from the control group.\n\nThe distribution of inserts for sample LWX777 from runs 22 (A) and 27+27_2 (B). The X-axis represents the number of reads, the Y-axis represents an insert size. The diagrams were obtained using Picard CollectIsertSizeMetrics v2.22.4.\n\nThe ratio of reads from sample LWX777 with the insert size exceeding 1000 nucleotides was 0.003% in case of the data combined from the different runs, while it was 0.005% in case of the previous sequencing without read integration. The obtained data imply that read merging was correct.\n\n\nConclusion\n\nIt is possible to use a sequence cartridge after 35-hour storage at +4°C, although the quality of the obtained data is reduced.\n\nMerging sequencing data can be successfully performed if the information about the localization in flow cells is saved in a read header.\n\n\nData availability\n\nRaw data for the sample LWX777 from runs 22 and 27_1+27_2 available at Sequence Read Archive (SRA), BioProject ID PRJNA683755: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA683755/\n\nLWX777_run27_2_united (SRA: SRS7871577) is an example of reconstructed fastq file from the 2 parts of interrupted run 27. First 139 nucleotides were received form run_name_L04_read.fq.gz fastq file with non-demultiplexed left reads for lane 4. LWX777_run22 (SRA: SRS7871575) is fastq files from previous run 22.\n\nZenodo: genomecenter/runcer-necromancer: Runcer Necromancer updated release (December 2020), http://doi.org/10.5281/zenodo.434035010.\n\nThis project contains the following extended data:\n\nFile S1. Summary report for run 27 lane 1 from MGISEQ-2000\n\nFile S2. Summary report for run 27_2 lane 1 from MGISEQ-2000\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\nScript available from: https://github.com/genomecenter/runcer-necromancer\n\nArchived script as at time of publication: http://doi.org/10.5281/zenodo.434035010.\n\nLicense: MIT", "appendix": "References\n\nMGI Tech official site. (Accessed on 14 September 2020). Reference Source\n\nFehlmann T, Reinheimer S, Geng C, et al.: cPAS-based sequencing on the BGISEQ-500 to explore small non-coding RNAs. Clin Epigenetics. 2016; 8(1): 123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeon SA, Park JL, Kim JH, et al.: Comparison of the MGISEQ-2000 and Illumina HiSeq 4000 sequencing platforms for RNA sequencing. Genomics Inform. 2019; 17(3): e32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenabouth A, Andersen S, Shi Q, et al.: Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing. NAR Genom Bioinform. 2020; 2(2): lqaa034. Publisher Full Text\n\nKorostin D, Kulemin N, Naumov V, et al.: Comparative analysis of novel MGISEQ-2000 sequencing platform vs Illumina HiSeq 2500 for whole-genome sequencing. PLoS One. 2020; 15(3): e0230301. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen J, Li X, Zhong H, et al.: Systematic comparison of germline variant calling pipelines cross multiple next-generation sequencers. Sci Rep. 2019; 9(1): 9345. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMGI Tech official site. (Accessed on 14 September 2020). Reference Source\n\nHuang J, Liang X, Xuan Y, et al.: A reference human genome dataset of the BGISEQ-500 sequencer. GigaScience. 2017; 6(5): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMGI Tech official site. (Accessed on 14 September 2020). Reference Source\n\nPavlova A: genomecenter/runcer-necromancer: Runcer Necromancer updated release (December 2020) (Version v1.0.1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4340350" }	[ { "id": "82441", "date": "08 Apr 2021", "name": "Simon Andrews", "expertise": [ "Reviewer Expertise bioinformatics", "ngs" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 example of being able to rescue data from a technically failed run by understanding the processing pipeline well enough to be able to adapt it to your needs. This information will be useful for others who end up in similar situations and may be able to adapt the methods or code presented here to their own situation. I'm somewhat surprised that the indexing of the sequences can be preserved even if the flowcell is moved, and it wasn't entirely clear whether that had actually happened in this run. The results presented make a convincing case that the data was indeed correctly re-paired. I would also have been interested to see the stats on the separation of the barcodes at the end of the run to see whether there was a higher proportion of miscalled bases within those sequences.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "98664", "date": "22 Nov 2021", "name": "Sergey Knyazev", "expertise": [ "Reviewer Expertise bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes the successful attempt of recovery of the genomic sequencing data after a power outage in the middle of a sequencing experiment. As a result of the outage, the sequencing machine stopped working prematurely that hindered the sequencing experiment. The manufacturer recommended throwing out the results of the entire experiment and starting another run. The article describes the methodology on how to overcome these recommendations and reconstruct results from both parts of the experiment before and after the outage. To reconstruct the experiment before the outage, the article proposes to use already generated files in a raw format that can be extracted from the machine. To reconstruct the experiment after the outage, the article proposes to run another experiment on the same cartridge with the same chemistry that was in the machine during the outage.\nWhile the idea looks interesting, the results and the conclusion are based on misleading assumptions. To prove that the restoration of the experiment was successful, the article tries to use quality control metrics that are designed for normal sequencing runs. The sequencing protocol that the article describes significantly diverges from the standard one because there is an outage in the middle of the experiment and the chemistry may be expired because of the delayed rerun. All that should be described in the article and all the assumptions should be explicitly discussed. Otherwise, the experiment requires rigorous case/control studies with running the sequencing machine in different outage settings with the comparing the results of the experiments with the outage with the experiments without the outage. In this case, at least two experiments are required, for example, one without protocol violation, and one after an outage on the same sequencing library.\nHaving said that, I recommend the article just simply describe all the limitations of its methodology very explicitly just to keep the audience from potentially misleading conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "7739", "date": "24 Jan 2022", "name": "Vera Belova", "role": "Author Response", "response": "We would like to thank Sergey Knyazev for the careful reading of our short report. We appreciate the time and effort that you have dedicated to providing your suggestions. We agree with Sergey’s general comment. For sure our short report contains a description of the method of merging reads for the MGI platform that we applied in a force majeure situation and it does not fit into the standard experiment. More explanation has been added in the conclusion section, we encourage readers to evaluate data quality on reference samples in this kind of situation. We hope that you find our response satisfactory." } ] } ]	1	https://f1000research.com/articles/10-22
https://f1000research.com/articles/10-849/v2	07 Sep 21	{ "type": "Brief Report", "title": "Comparing carbon emissions between online and in-person study for a cohort of overseas students: A retrospective cohort study", "authors": [ "Richard F Heller", "Ya-Yen Sun", "Zhe Guo", "Arunima Malik", "Ya-Yen Sun", "Zhe Guo", "Arunima Malik" ], "abstract": "Background: One of the benefits of online education is the potential reduction in carbon emissions through the decrease in travel to attend a university in person. We estimated the savings in CO2 emissions of an international cohort of master’s students who studied fully online from their home countries, rather than travelling to the UK and living there while attending university. Methods: The city and country of residence of a cohort of students who first enrolled in the fully online Peoples-uni/Manchester Metropolitan University MPH programme between the second semester of 2011 and the first semester 2013 were recorded. We estimated the aviation emissions between Manchester, UK and the cities where students reside, and subtracted the per capita emissions for the country of origin from the per capita emissions for the UK over the time that the student would have spent in Manchester as a full-time student, based on the semester in which they first enrolled. Results: 128 students enrolled from 55 cities in 31 countries. 93 students were from a range of African countries and 18 from the Indian sub-continent. Flights to and from Manchester were estimated to have accounted for 114,553kg of CO2 and living in Manchester for the duration of their course compared with staying in the home country would have been equivalent to 854,904kg of CO2. The combined net savings was 969,457kg of CO2. Conclusions: A small cohort of overseas students, largely from Africa and India, studied online rather than attending university in the UK. The saving by this small cohort of nearly a million kg of CO2 emissions through not attending university in-person offers an indication of the potential environmental benefits of offering university education online to overseas students.", "keywords": [ "Online learning", "carbon emissions", "airline travel", "overseas students" ], "content": "Background\n\nOne of the benefits of online education is the potential reduction in carbon emissions through decreasing travel to attend university in person. However, there is little evidence in the existing literature that quantifies this potential for overseas students, but one study concludes: “The introduction of online education allows [...] a huge reduction in carbon emissions and could thus help HEIs [Higher Education Institutions] to achieve their energy efficiency and sustainability goals”1. Other studies have examined the carbon footprint of universities, with one finding that travel by overseas students to the university accounted for 6% of total emissions2 and another, while describing a large variation in estimated carbon emissions between different universities in Texas, also estimated the carbon emissions from travel through a ‘study abroad’ programme in one university3.\n\nPeoples-uni, a volunteer led educational charity, provided fully online master’s level courses to health professionals in low- to middle-income countries (LMICs) from 2008 to 20214. For four semesters between 2011 and 2013, a partnership allowed students to enrol in the Master of Public Health (MPH) offered by Manchester Metropolitan University (MMU) by solely online study through the Peoples-uni without travel to the UK.\n\nThis paper estimates the savings in CO2 emissions by this cohort of students who studied fully online from their home countries rather than travelling to and living in Manchester to attend the University in-person.\n\n\nMethods\n\nA retrospective cohort study explored the records in the Peoples-uni database of each of the students who first enrolled through Peoples-uni in the MMU MPH award programme between the second semester of 2011 and the first semester of 2013. The city and country of residence were recorded, as was the final award gained. Even though the course was part-time, we assume that students would have been living in Manchester full-time and would have travelled by air from their home city. We assumed that they would have lived in Manchester for 18 months to complete a full 180 credit MPH, 12 months for those exiting with a 120 credit Graduate Diploma (passing all coursework except for the Dissertation) or 6 months for those exiting with a 60 credit Graduate Certificate (passing half the number of modules required for the Graduate Diploma). For students who passed some modules, but not enough to earn a Graduate Certificate, we assumed they would have spent 3 months in Manchester, and for those who passed no modules we assumed they would have withdrawn before travelling to Manchester. The dataset for this report can be found here5.\n\nThe differences of carbon emissions during participation in the MMU MPH programme are calculated as the following:\n\nNet emissions = (emissions of living in Manchester) – (emissions of living at home country) + round trip air transport emissions\n\nIf net emissions are larger than zero, this implies the online MMU MPH programme creates an environmental benefit - with a carbon footprint at home smaller than the footprint when living in Manchester combined with the air travel.\n\nTo calculate the difference, we first used the International Civil Aviation Organization (ICAO) carbon emissions calculator6. The ICAO provides the comprehensive city-pair carbon dioxide emissions from air travel by taking into account aircraft types, route specific data, passenger load factors and cargo carried. We estimated the aviation emissions between Manchester, UK and the city where students resided. To avoid overestimating the environmental impact of the travel, we took a conservative approach by choosing the route with fewest number of stops and lowest flight time or miles where this was an option, even though these may not have been the cheapest options, nor the actual flights used by the students. Road travel from a city without an international airport was recorded but not included in a calculation of emissions as the mode of travel was unknown and the estimates would have been imprecise.\n\nTo calculate the emissions of students living in the UK compared with their home country, the annual per capita CO2 emissions for each country were taken for the relevant years from data collected by the Carbon Dioxide Information Analysis Center and reported in OurWorldInData.org7. The per capita emissions for the country of origin were subtracted from the per capita emissions for the UK over the time that the student would have spent in Manchester as a full-time student, starting with the semester in which they first enrolled.\n\n\nEthics statement\n\nAs part of the application process for entry to Peoples-uni courses, students were informed that their anonymised information might be used for research into the outcomes of the education programme. Data from the Peoples-uni database were extracted by one of the researchers (RFH) and de-identified by deleting the names of the students from the resulting spreadsheet shared for analysis with the other authors, and for the resulting publication. No ethical approval was sought due to the low-risk nature of the study.\n\n\nResults\n\nFrom 2011 to 2013, 128 students enrolled in the MMU MPH programme from 55 cities in 31 countries, 93 students were from Africa and 18 from the Indian sub-continent.\n\n94 students gained an MPH, from which we recorded an assumed 18 months living in Manchester, 9 gained a Graduate Diploma, equating to 12 months in Manchester, and 16 students gained a Graduate Certificate, equating to 6 months in Manchester. 5 students passed two modules, corresponding to 3 months in Manchester, and 4 students gained no passes and are assumed not to have travelled to Manchester at all.\n\n35 students started in the second semester of 2011, 24 and 22 respectively in the first and second semesters 2012, and 47 in the first semester of 2013. Although all students were from LMICs, some were living in high-income countries at the start of their studies.\n\nTwo students started the MPH programme in the UK, so were not counted in the calculation of transport emissions. Flights to and from Manchester were estimated to have accounted for 114, 553 kg of CO2 emissions, with an average of 924 kg per student. Transport emissions are largely determined by distance, and the largest emissions on flights were those flying intercontinental from Fiji (2,133 kg), Papua New Guinea (1,635 kg) and Zimbabwe (1,495 kg) to Manchester. Figure 1 shows the emissions for each country – where students came from more than one city in a country these were averaged to show country data.\n\nX axis shows the country from which the students come – where students come from more than one city in the country, the mean has been calculated to characterise the country. Y axis shows kg CO2.\n\nThe two students who enrolled from the UK had no change in emissions, and seven students came from countries (South Africa, USA, Canada and United Arab Emirates) with higher emissions than in the UK, so contributed negative counts. Overall, the emissions per capita are linked strongly to national economic development status – the higher the wealth the larger the emission footprint. Because the MMU MPH programme was mainly offered to students from LMICs, students’ carbon footprint in their home country is generally lower than it would be living in Manchester, although this will vary over time. As examples, the net CO2 emission estimates used for 2013 were 7,354 kg for Manchester, 103 kg for Ethiopia and 72 for Rwanda. For the group as a whole, living in Manchester for the duration of their course compared with staying in the home country would have been equivalent to a net excess of 854,904 kg of CO2.\n\nCombining transport and living gives an estimate of total excess net emissions of 969,457 kg of CO2. Figure 2 shows the total net emissions per country.\n\nX axis shows the country from which the students come. Y axis shows the net difference in emissions between the UK and each country in kg CO2.\n\n\nDiscussion\n\nThis cohort of 128 master’s students was estimated to have saved 969,457 kg of CO2 through studying online from their home country rather than travelling to and living in Manchester, UK to attend in person.\n\nWe used conservative assumptions for flight estimations in terms of number of stops and routes taken, and also assumed that the students travelled alone without family and did not return home during the programme. Flight emissions may reduce over time with increasing global attention to the climate change issue. Per capita emissions will also change over time in different ways across countries.\n\nWe have assumed that a student living in Manchester would have the same consumption patterns as the general population, and so created our method of calculating their consumption by subtracting the per capita CO2 emissions of their own country from that of the UK. It may be that students have lower consumption patterns than the general population, although the university campus has a high carbon footprint8. Future research could consider specific supply chain aspects for quantifying reductions in emissions from online learning. To this end, a global multi-regional input-output model could be integrated with detailed information on expenditure patterns of students on a country-by-country basis for quantifying their at-home carbon footprint, and the footprint if they travelled to the UK. Such an analysis could be performed at a sector-level, enabling the quantification of hotspots. Future work could also focus on expanding such an assessment to university-wide quantification of emission savings from online learning, beyond the assessed master’s programme in this work. A university-wide assessment could also include savings through online working of the teaching staff, a possible decrease in electricity consumption in lecture theatres (and possible increase from students’ perspective). Future research could also consider the costs associated with the sourcing of equipment for accessing online material, such as laptops, internet plans, and associated carbon emissions.\n\n\nConclusion\n\nAlthough there have been a number of studies discussing the benefits of online learning on carbon emissions, the full contributions of overseas students to emissions have not yet been explored. Caird et al.9 calculated that among 15 higher education institutions in the UK, distance-based education models achieved an 83% reduction in carbon emissions, with the fully online model achieving the lowest carbon emissions. Similarly, Perales Jarillo et al.10 have set the benefits of online education in the context of Sustainability Development Goals.\n\nProject Atlas, quoting UNESCO data, estimated in their 2020 report that there were more than 5.6 million higher education students globally that were studying abroad11. In each of the top three countries receiving overseas students, the United States, the United Kingdom and Canada, more than 20% of all students were international. In the UK in 2019/20 there were more than 250,000 postgraduate non-UK students, the majority from outside the EU12. Considering that the countries from which most overseas students come from have lower emissions per capita, having international students enrolled in in-person programmes will create a net emission increase compared with online-study. Given the large number of overseas students globally, their impact on carbon emissions is considerable.\n\nThat even a small cohort of international students, largely from Africa and India, studying online rather than travelling to the UK likely saved nearly a million kg of CO2 provides an indication of the extent of the savings that could be made through the development of online education for overseas students.\n\n\nData availability\n\nData come from International Civil Aviation Organization (ICAO) carbon emissions calculator6 https://www.icao.int/environmental-protection/CarbonOffset/Pages/default.aspx; and CO2 and Greenhouse Gas Emissions7 https://ourworldindata.org/co2-and-other-greenhouse-gas-emissions\n\nZenodo: Saving carbon emissions through online learning for overseas students [Data set] https://doi.org/10.5281/zenodo.53358665\n\nThis project contains the following underlying data:\n\n- Student data and calculations, and World Bank emissions data\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nVersteijlen M, Salgado FP, Groesbeek MJ, et al.: Pros and cons of online education as a measure to reduce carbon emissions in higher education in the Netherlands. Curr Opin Environ Sustain. 2017; 28: 80–89. Publisher Full Text\n\nOzawa-Meida L, Brockway P, Letten K, et al.: Measuring carbon performance in a UK University through a consumption-based carbon footprint: De Montfort University case study. J Clean Prod. 2013; 56: 185–198. Publisher Full Text\n\nBailey G, LaPoint T: Comparing Greenhouse Gas Emissions across Texas Universities. Sustainability. 2016; 8(1): 80. Publisher Full Text\n\nHeller RF, Strobl J, Madhok R: Online Education for Public Health Capacity Building in Low- to Middle-Income Countries: The Peoples-uni Experience. IRRODL. 2019; 20(1). Publisher Full Text\n\nHeller RF: Saving carbon emissions through online learning for overseas students [Data set]. Zenodo. http://www.doi.org/10.5281/zenodo.5335866\n\nInternational Civil Aviation Organization (ICAO) carbon emissions calculator. Reference Source\n\nRitchie H, Roser M: CO2 and Greenhouse Gas Emissions. 2020. Reference Source\n\nYañez P, Sinha A, Vásquez M: Carbon Footprint Estimation in a University Campus: Evaluation and Insights. Sustainability. 2020; 12(1): 181. Publisher Full Text\n\nCaird S, Lane A, Swithenby E, et al.: Design of higher education teaching models and carbon impacts. Int J Sust Higher Ed. 2015; 16(1): 96–111. Publisher Full Text\n\nPerales Jarillo M, Pedraza L, Moreno Ger P, et al.: Challenges of Online Higher Education in the Face of the Sustainability Objectives of the United Nations: Carbon Footprint, Accessibility and Social Inclusion. Sustainability. 2019; 11(20): 5580. Publisher Full Text\n\nInstitute of International Education: Project Atlas. 2021. Reference Source\n\nHESA, the Higher Education Statistics Agency: Higher Education Student Statistics: UK, 2019/20 - Where students come from and go to study. Reference Source" }	[ { "id": "93064", "date": "14 Sep 2021", "name": "Vincent Tawiah", "expertise": [ "Reviewer Expertise 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\nThis paper addresses an interesting and relevant topic on how online education could solve environmental problems. I suggest the authors improve the paper by considering the following points:\nGiven that online education covers international activity, I suggest that the authors provide more literature on how international activities affect the environment and then narrow it down to online education. The authors could consider the following papers:\nThe environmental footprint of China-Africa engagement by Tawiah et al. (2020)1; Energy resource melioration and CO2 emissions in China and Nigeria; Efficiency and trade perspective by Li et al. (2020)2.\n\nPolicy implication: COVID-19 has forced many HEI to move online; hence, I expect the authors to provide some policy implications on findings nested with the COVID-19 situation. One way is to articulate how online education could reduce emissions and make your studies more relevant in this COVID era.\nAll the best.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7174", "date": "24 Sep 2021", "name": "Richard F Heller", "role": "Author Response", "response": "Reviewer Report Vincent Tawiah, DCU Business School, Dublin City University, Dublin, Ireland This paper addresses an interesting and relevant topic on how online education could solve environmental problems. I suggest the authors improve the paper by considering the following points: Given that online education covers international activity, I suggest that the authors provide more literature on how international activities affect the environment and then narrow it down to online education. The authors could consider the following papers: The environmental footprint of China-Africa engagement by Tawiah et al. (2020)1; Energy resource melioration and CO2 emissions in China and Nigeria; Efficiency and trade perspective by Li et al. (2020)2. Author response: We have added both of these references, and added the following to the Conclusion: “There is a literature on the way in which numerous international economic activities affect the environment 13,14 and the importance of international education to the economy of many countries demonstrates the value of considering how online education might contribute to a reduction in global CO2 emissions. ” Policy implication: COVID-19 has forced many HEI to move online; hence, I expect the authors to provide some policy implications on findings nested with the COVID-19 situation. One way is to articulate how online education could reduce emissions and make your studies more relevant in this COVID era. Author response: We have added the following to the Conclusion: “The benefits of reducing CO2 emissions through online education for international students should be seen in the context of the COVID era, which has demonstrated the importance of online education and the limits to international travel.” All the best. Author response: Many thanks for your helpful suggestions. We hope that the corrections we have made capture your points." } ] }, { "id": "93060", "date": "15 Sep 2021", "name": "Andy Lane", "expertise": [ "Reviewer Expertise Environmental systems", "innovation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe introduction is quite poor in setting the context. Some material in the conclusions should be in the introduction, for example, the discussion of Caird S, Lane A, Swithenby E, et al.: Design of higher education teaching models and carbon impacts. Int J Sust Higher Ed. 2015; 16(1): 96–1111 would help with that context rather than being an afterthought in the conclusions.\nThe method used in this study is very high level and crude. It ignores the HE context (in terms of HE systems, climate action and sustainability policies and GHG emissions data) and does not describe the two teaching models compared; so consequently, it offers no insights into systemic aspects of HE institutional systems and teaching models. In other words, the method would be no different if it were calculating the effects of visiting the UK on holidays or for work for the same duration, and so it offers limited insight from a HE perspective. The title is therefore misleading because it does not look at the impacts of ‘online and in-person study’, instead, it estimates the impact of air travel of overseas students.\nAs far as we can tell, limited primary data was collected, other than on the number of students on a programme, their nationality, and their qualification programme. The method does not take student or staff activities related to teaching and learning into account, other than to make assumptions on average behaviours rather than any self-reporting of 'actual' behaviours. The study assumes:\nstudents living in Manchester would have the same consumption patterns as the general population in the UK (is there no variation in consumption within countries?);\n\nstudents would have been living in Manchester full-time;\n\nthe length of time students would have lived in Manchester, based on the time needed to complete their qualification programmes;\n\nstudents would have travelled by air from their home city;\n\nflight impacts in terms of the number of stops and routes taken;\n\nstudents travelled alone without family and did not return home during the programme.\nThese limitations should be acknowledged, i.e. the study does not consider HE systems nor students specifically, and so the study is just an estimation of travel emissions related to visitors to the UK for a specific period of time.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7175", "date": "24 Sep 2021", "name": "Richard F Heller", "role": "Author Response", "response": "Reviewer Report Andy Lane, The Open University, Milton Keynes, UK Sally Caird, The Open University, Milton Keynes, UK The introduction is quite poor in setting the context. Some material in the conclusions should be in the introduction, for example, the discussion of Caird S, Lane A, Swithenby E, et al.: Design of higher education teaching models and carbon impacts. Int J Sust Higher Ed. 2015; 16(1): 96–1111 would help with that context rather than being an afterthought in the conclusions. Author response: We have quoted this paper at the start of the Background which now reads as follows: “One of the benefits of online education is the potential reduction in carbon emissions through decreasing travel to attend university in person. Caird et al. 1 calculated that among 15 higher education institutions in the UK, distance-based education models achieved an 83% reduction in carbon emissions, with the fully online model achieving the lowest carbon emissions. Estimates included travel, purchase and use of ICT devices, purchase of books and publications and use of paper for printing, residential and home energy use and campus site operations. Versteijlen et al. conclude: “The introduction of online education allows [...] a huge reduction in carbon emissions and could thus help HEIs [Higher Education Institutions] to achieve their energy efficiency and sustainability goals” 2” The method used in this study is very high level and crude. It ignores the HE context (in terms of HE systems, climate action and sustainability policies and GHG emissions data) and does not describe the two teaching models compared; so consequently, it offers no insights into systemic aspects of HE institutional systems and teaching models. In other words, the method would be no different if it were calculating the effects of visiting the UK on holidays or for work for the same duration, and so it offers limited insight from a HE perspective. The title is therefore misleading because it does not look at the impacts of ‘online and in-person study’, instead, it estimates the impact of air travel of overseas students. Author response: To reflect this comment we have changed the title as follows: “Impact on carbon emissions of online study for a cohort of overseas students: A retrospective cohort study”. We have also added to the Methods the following: “We did not estimate the carbon emissions associated with different educational processes themselves.” We have also added the following to the Discussion: “Caird et al. 1 estimate 36kg CO2 per 100 study hours for UK based fully online courses (compared with 278kg for face-to-face teaching). Applying this estimate for online teaching to our cohort would equate to 648kg over the course of the master’s degree, and 68,796kg for the whole cohort. However, it is difficult to apply this to Peoples-uni which did not have a campus, used Open Educational Resources and whose students live in LMICs.” We have also added to our original sentence in the Discussion “Future work could also focus on expanding such an assessment to university-wide quantification of emission savings from online learning, beyond the assessed master’s programme in this work.” which now reads: “Future work could also focus on expanding such an assessment to university-wide quantification of emission savings from online learning, beyond the assessed master’s programme in this work, to provide an accurate estimate of the emissions from different teaching models related to the ‘export’ of higher education to LMIC populations.” We have also changed the final sentence in the Abstract from “The saving by this small cohort of nearly a million kg of CO 2 emissions through not attending university in person offers an indication of the potential environmental benefits of offering university education online to overseas students.” to “The likely saving by this small cohort of nearly a million kg of CO 2 emissions offers an indication of the potential environmental benefits of offering university education online to overseas students.” As far as we can tell, limited primary data was collected, other than on the number of students on a programme, their nationality, and their qualification programme. The method does not take student or staff activities related to teaching and learning into account, other than to make assumptions on average behaviours rather than any self-reporting of 'actual' behaviours. The study assumes: students living in Manchester would have the same consumption patterns as the general population in the UK (is there no variation in consumption within countries?); students would have been living in Manchester full-time; the length of time students would have lived in Manchester, based on the time needed to complete their qualification programmes; students would have travelled by air from their home city; flight impacts in terms of the number of stops and routes taken; students travelled alone without family and did not return home during the programme. These limitations should be acknowledged, i.e. the study does not consider HE systems nor students specifically, and so the study is just an estimation of travel emissions related to visitors to the UK for a specific period of time. Author response: We thank the Reviewers for these suggestions which clarify the nature of the study. As indicated above, we have now acknowledged the limitations of the study at various points in the paper, including in the title, and feel that the changes more accurately reflect the nature of the study and any implications." } ] } ]	2	https://f1000research.com/articles/10-849
https://f1000research.com/articles/11-182/v1	14 Feb 22	{ "type": "Research Article", "title": "Colorectal cancer on a dish: exploring the 3D-sphere culture of primary colorectal cancer cells from an Indonesian perspective", "authors": [ "Murdani Abdullah", "DR Noor", "Amanda Pitarini Utari", "Virly Nanda Muzellina", "Nur Rahadiani", "Radiana Dhewayani Antarianto", "DR Noor", "Amanda Pitarini Utari", "Virly Nanda Muzellina", "Nur Rahadiani", "Radiana Dhewayani Antarianto" ], "abstract": "Background : Colorectal Cancer (CRC) is one of the deadliest types of cancer and has emerged as one of Indonesia's most devastating diseases. The growing number of colorectal cancer cases is frequently undiagnosed until the disease has progressed to a metastatic stage. This issue has lasted for years, limiting therapy options and resulting in a bad prognosis for the majority of patients. Thus, the purpose of this work is to develop a CRC detection method for Indonesia and other low-middle income nations that integrates in vitro 3D culture, molecular analysis, and in silico analysis. Methods : Colorectal cancer biopsies were transported to the lab and underwent mechanical disaggregation and centrifuged at 300 x g for five minutes. Approximately 10,000 cells were seeded in each Nunc-Sphera 96-well plate (u-bottom) for the following 7 days in standard culture medium. The 3D-sphere was harvested and RNA was extracted afterwards. Molecular analysis was performed using qPCR and the Human Cancer Pathway Profiler. Protein interaction and pathway analysis were conducted using STRING and Reactome online tools. Results : Following initial seeding, primary CRC 3D-spheres were grown for 14–16 days. Gene profiling and in silico analyses suggest that CDC20, AURKA, and ACLY are expressed at lower levels than the positive control in the 3D-sphere. These markers have been implicated in metastasis, CRC proliferation, and as a drug target ligand. Conclusion : A combination of 3D culture, gene profiling, and in silico analysis is feasible to detect CRC for Indonesia and other low- and middle-income countries. A future possibility is to use minicolorectal cancer in a dish for ex vivo cancer modeling and pharmacological testing.", "keywords": [ "Colorectal cancer", "3D-sphere", "primary cell culture", "in silico analysis", "CRC detection." ], "content": "Introduction\n\nColorectal cancer has been found as one of the deadliest cancers worldwide and may be affected by various causes, including lifestyles, smoking habits, and western food diets. Based on the Global Cancer Observatory (GLOBOCAN), colorectal cancer is the third most common cancer with 9,000 deaths and 15,000 new cases in 2018 worldwide (Bray et al., 2018). So far, cancers have been commonly treated using anticancer, targeted chemotherapy, or particular molecular markers that are overexpressed in cancer patients (Perry & Weitzman, 2005; Kumar et al., 2017). Such progress was mainly affected by the successful breakthrough of cancer modelling, which then led to a greater connection between laboratory invention and clinical implications (Thomas et al., 2016). Therefore, cancer modelling is one of the keys to the development of therapeutic agents, while identification and validation of molecular markers are needed, respectively (Perry & Weitzman, 2005; Thomas et al., 2016).\n\nOne of the most common cancer models, in particular preclinical models, is cancer cell lines, which are pure cancer populations that are derived from cancer patients. This model has been widely used in cancer biology and also the development of anticancer drugs worldwide (Ferreira, 2013; Mirabelli, Coppola & Salvatore, 2019). Cancer cell lines were mostly well characterized in many various levels, from genetics to expression systems, including transcriptomic and proteomics characterizations (Mirabelli, Coppola & Salvatore, 2019). However, since tumors were constructed with various types of cells, cancer cell lines were lack of tumor heterogeneity such as stromal cells, and do not represent the original tumors (Ferreira, 2013; Strickaert et al., 2017). On the other hand, ex-vivo models are models that are derived from tumor tissue which may be given to animals as in vivo models or grown as in vitro models. These methods represent tumor characteristics and in vivo heterogeneity such as tumor microenvironment (Meijer et al., 2017). Moreover, ex-vivo models are not only potential for drug testing, but also models to predict patient response towards particular drugs (Meijer et al., 2017). Therefore, focusing on developing ex-vivo models is potential in many ways.\n\nRecently, there have been several types of ex-vivo models, from in vitro to in vivo models. in vivo models may represent the tumors, for instance, using Patient-Derived Xenograft (PDX) models to predict drug responses. However, ethical issues may arise while considering using animal models in cancer research (Festing & Wilkinson, 2007; Meijer et al., 2017). On the other hand, in vitro models such as cancer cell lines do not always require ethical clearance for each experiment. Nevertheless, in vitro models lacked tumor heterogeneity, which may have later related to an unfamiliar response to the patients (Ferreira, 2013; Strickaert et al., 2017). Respectively, primary cancer cell cultures derived from patients have been acknowledged to mimic tumors in vivo and as potential models (Miserocchi et al., 2017).\n\nPrimary ex vivo models may become suitable (Miserocchi et al., 2017). However, isolation and establishment of primary cell cultures from cancer patients, in particular colorectal cancer, is found to be challenging (Failli et al., 2009). Thus, in this study, we were shown fast and simple methods to dissociate tumor biopsies for primary culture. We also identified similar expressions of three genes: ACLY, Aurora Kinase A (AURKA), and CDC20 in both ex-vivo primary cell culture models. These potential tools predict genes that are expressed while the cells grow in an in vitro environment.\n\n\nMethods\n\nThe study has been approved by the Ethical Committee of Faculty of Medicine, Universitas Indonesia (Protocol ID: 20-04-0643, version 02, June 29th, 2020). Recruited subjects provided their written informed consent for biopsies donation (biobanking) and for the use and publication of their data for research purposes. All participants signed an informed consent form before participating in the study.\n\nBiopsies were cleaned with 0.1 percent Povidone Iodine in a complete primary culture medium (DMEM) including 10% FBS and 5% antibiotic-antimycotic solutions at Biosafety Cabinet Level 2. This procedure proved crucial for successfully diagnosing primary colorectal cancer and preventing contamination by washing it with solutions containing high concentrations of antibiotics and antimycotics. Following the washing process, the biopsy was rinsed in full medium 150 mm Petri plates (Biologix, Germany) to get fragments with a diameter of 1-3 mm3. Then, fragments were pinched with sterile tweezers and scraped with a cell scraper to detach the cells from their associated tissue. After that, 5 ml complete medium was added to the fragment-containing plate and numerous times resuspended with 10 ml serological pipette tips before being passed to pre-wet 100 m cell strainers on 50 ml centrifuge tubes. Finally, the leftover pieces on the cell strain were mashed using a 12 ml syringe plunger. The medium was added to the cell top to allow the cells to flow through. After centrifuging the suspension at 300 × g for 5 minutes, the pellets were resuspended in complete mediums.\n\n3D-sphere primary CRC was harvested on the day of 7. Approximately 10,000 cells were obtained by pipping up and down to a single cell suspension. The suspension was transferred to 1.5 ml of centrifuge tubes and spun at 300 × g for five minutes afterward. After removing the supernatant, the cells were resuspended in 100 l of DNA/RNA shield (ZymoResearch, USA). The RNA was extracted using RNA Miniprep Plus (ZymoResearch, USA) according to the manufacturer’s protocols. The RNA purity and concentrations were measured using nano spectrophotometers.\n\nRNA was reverse transcribed to cDNA using the RT2 First-strand kit (Qiagen, Germany #Cat#330401) according to the manufacturer’s protocols. The cDNA concentration and purity were measured afterward using nano spectrophotometers.\n\nSamples cDNA were processed to qPCR assay according to manufacturer’s instructions of RT2 Profilers (Qiagen, Hildenburg, Germany, PN.330231, Cat#PAHS-033ZC-6). qPCR was conducted using standard PCR conditions as follows: 1 cycle of 10 min, 95°C HotStart DNA Taq Polymerase is activated by this heating step, continued by 40 cycles of fluoroscence data collection in 15 s in 95°C and 1 minute in 60°C. The qPCR assay was performed in ABI 7500FAST machine (Applied Biosystem, USA). Lists of housekeeping genes were already included on the kit and the primers were already designed by Qiagen.\n\nProtein-protein from positively expressed genes was analyzed using the String-8 Platform using multiple protein list, interactive was set to experiment, gene fusion and co-expression and highest confidence (0.9oo) developed by Jensen et al. (2009). The PPI was enriched one time by clicking “more” and represented as images.\n\n\nResults\n\nPrimary cultures were developed from two patients who had undergone surgical resections. Patients' profiles were then monitored from the Electronic Health Record of dr. Cipto Mangunkusumo Hospital, Jakarta. The CRC 001 was developed in a 57-year-old female. The tumor locations were rectal, the stadium was IV, the tumor size was T4b, N1 nodules, liver metastasis, and a well-differentiated adenocarcinoma. The CRC 002 was developed in a 45-year old male. The tumor location was the rectum. The stadium was II, N0 nodules, no metastasis, and also a well differentiated adenocarcinoma. The sample clinicopathology profiles are presented in Table 1.\n\nEndoscopic and resected tissues were sampled, and the samples were transferred into a transport medium and delivered within 24 hours at room temperature. For resected tissues, since the samples were large, after mechanical disaggregation, viability using a trypan blue exclusion assay should be conducted to determine appropriate cell numbers that should be seeded into the well. Therefore, we seeded the samples with 10,000 cells per well to demonstrate cell morphology. Cell morphology was evaluated by visualizing the samples using an Axio Carl Zeiss inverted microscope in Axiovision Zen Blue 2.1 edition.\n\nWe grew the cells in standard media in an ultra-low u-shaped NuncSphera plate (Nunc, Thermoscientific, USA). Three–seven days after initial seeding, 3D cultures were created and captured using Carl Zeiss Microscopy, Zen Blue Editions software at 5× magnification. The majority of cells were cultured in aggregates. However, the cells were not developed into circular shapes in a regular manner. The heterogeneity of cells produced from single biopsies and their ability to develop aggregated were proven to be dependent on the biopsies' intracellular composition. Additionally, our models demonstrated significant patient characteristics, as illustrated in Figures 1 and 2.\n\nPrimary cultures are 3 days after initial seeding. Cells were captured using Carl Zeiss inverted microscopy at 20 × magnification. All of the samples were derived from adenocarcinoma patients, yet distinct morphology and growth patterns were observed.\n\nAfter 14 days, primary cultures cells were captured using Carl Zeiss inverted microscopy at 10 × magnification. All of the samples were derived from adenocarcinoma patients, yet distinct morphology and growth patterns were observed.\n\nA pathway finder was used to investigate human cancer pathways, while positive expressing cells were analyzed. The CT was positioned above the signal in accordance with protocol specifications. Amplification of samples utilizing RT-Profilers using this pathway resulted in the expression of three genes, namely ACLY, AURKA, and CDC20, as indicated in Table 2. On CRC 001, the genes were largely expressed earlier than on CRC 002. However, because the data were not standardized for undiscovered housekeeping gene levels in both samples, such values were not evaluated, despite the presence of positive and reverse transcript controls. Those undetected may be due to the assay using insufficient numbers of cells (10,000 cells), which may be implied by undetected expressions. On the other hand, detected samples may be associated with elevated levels of gene expression, resulting in the detection of the genes.\n\nThere were two important pathways at this point, one involving metabolism-related genes, ACLY, and another involving cell cycle-related genes, AURKA and CDC20, respectively. As a result, this may be studied individually based on the intricacy of one pathway versus another. In Figure 3 we show that CDC20 and AURKA that has been enriched with other proteins from the STRING database, with strict criteria (highest confidence = 0.900), experiments, gene fusion, and co-expression. The co-expression score between CDC20 and AURKA was 0.808, while the experimental score was 0.487, and the combined score was 0.987 (Jensen et al., 2009). Biologically, the expression of both proteins may result in the breakdown of mitotic proteins, hence influencing chromatid segregation and mitotic protein degradation.\n\nCDC20 and AURKA was strongly connected (combined score = 0.987) while ACLY was not related to CDC20 and AURKA.\n\n\nDiscussion\n\nEx-vivo models can be created from patients in a variety of ways, including primary culture, xenografts, and organoids (Meijer et al., 2017) Primary cell culture was certainly a supply of cells for a variety of applications, including preclinical models and personalized cancer therapy (Meijer et al., 2017; Miserocchi et al., 2017). According to our study, one of the most suitable methods for application in clinical settings was primary cell culture from patients, which may be collected concurrently with biopsies. We used samples from two distinct types of colorectal cancer biopsy in this study: endoscopic biopsies and resection tissue acquired during surgery.\n\nOne point to emphasize prior to sample collection is the use of a complete medium containing high concentrations of antibiotic and antimycotic solutions, which we did by supplementing DMEM with 10% FBS and 5% Antibiotic Antimycotic solutions. Supplemented mediums with FBS were thought to reduce cell death in these conditions, but ABAM solutions avoided contamination. Because colon tissues may have a high concentration of gut microbiotas, we recommended a beginning concentration of 5%, consistent with earlier research (Jeppesen et al., 2017; Failli et al., 2009). Using antibiotics and antimycotics at high concentrations may limit cell development, whilst using them at low concentrations below 2%, for example, 1%, increases the susceptibility of samples to be contaminated by microbial organisms.\n\nPrimary culture development can be used in a variety of ways, including two-dimensional or three-dimensional culture systems (Miserocchi et al., 2017). We employed 3D rather than 2D in our investigation. When compared to two-dimensional systems, primary culture in three dimensions results in a more physiological environment because the cells are aggregated and have three-dimensional structures that may facilitate tumor core formation and more closely imitate in vivo systems (Miserocchi et al., 2017; Riffle & Hegde, 2017). According to Riffle et al., three-dimensional tumor models were representative. In our instance, three-dimensional media will allow us to cultivate spheroids for a minimum of three to seven days without medium modifications.\n\nWe proved that growing primary cultures from resected tissue was not difficult by considering the optimal transport medium previously indicated to minimize such contamination. Samples may be extracted in around two hours using our procedures. Appropriate seeding with 10,000 cells may be employed for 3–7 days. In Figures 1 and 2, we demonstrate successful formation from two different patients (Table 1). Following that, samples can be harvested and seeded with 1,000 cells per well, as illustrated in figure 7A. However, no statistically significant variations in cell growth capacity were identified (Figure 7b), but morphological differences were observed. In the future, our simple methods may be employed to establish primary cell culture in three-dimensional (3D) systems. Additional pharmacological testing or the development of preclinical models employing 3D culture systems may be pursued in the future.\n\nWe next used the Cancer Pathway Profiler to analyze the cancer pathways that were activated in both samples utilizing this spheroid culture. The results indicated that Aurora kinase A/AURKA (NM 003600), cell division cycle protein 20 homolog/CDC20 (NM 001255), and ATP-Citrate Lyase/ACLY (NM 001096) were all expressed positively. However, housekeeping genes such as Beta actin/ACTB (NM 001101) and Glyceraldehyde-3-phosphate dehydrogenase/GAPDH (NM 002046) were not found, although positive controls and reverse transcriptase were. One possibility is that the amount of cDNA included in the samples was insufficient, resulting in the absence of multiple genes, including the housekeeping genes. On the other side, the presence of AURKA, CDC20, and ACLY may imply that these genes were expressed at a higher rate than housekeeping genes.\n\nAURKA and CDC20 are important in cell cycle regulation in biological systems, whereas ACLY is a metabolic enzyme. AURKA is a serine-threonine kinase that catalyzes the activation of numerous proteins during mitosis (Koh et al., 2017). AURKA was a gene that induced anaphase during complex biological processes. These include the activation of spindle assembly checkpoints (SAC) and the reading of bipolar chromosome movements (Courthéoux et al., 2018). Increased AURKA expression in cancer cells may result in cells evading SAC, resulting in aneuploidy, and so inhibiting AURKA may be beneficial in future cancer treatment (Courthéoux et al., 2018). Increased AURKA expression was associated with a poor outcome in colorectal cancer liver metastases and may become a useful biomarker for prognosis prediction (Goos et al., 2013). Additionally, AURKA exhibited the potential to serve as a novel prognostic marker in colorectal cancer (Koh et al., 2017). Through co-regulation of Myc-driven oncogenes with TPX2, AURKA was demonstrated to increase carcinogenesis in colorectal tumors (Takahashi et al., 2015). Simultaneously, we generated ex vivo models from colorectal adenocarcinoma. Positive expression of AURKA may be essential for cells to develop in vitro.\n\nEx vivo models also expressed CDC20 similarly in our findings. These proteins are crucial for initiating anaphase by interacting with Anaphase-Promoting Complexes (APC/C). Due to the maintained CDC20 level, immature anaphase might be prevented (Mondal et al., 2007). In oral tumors, overexpression of CDC20 impairs aneuploidy (Mondal et al., 2007). TP53 and TP21 interactions negatively controlled CDC20 expression via CDH-CHE motifs in CDC20 promoters. Kidokoro et al. demonstrated that silencing CDC20 mRNA results in G2/M arrest and cell growth retardation (Kidokoro et al., 2008, p. 20). Thus, positive expression of CDC20 in our ex vivo models may demonstrate that CDC20 is required for tumor growth to continue. CDC20 and AURKA were both shown to be associated with cell cycle progression and may be related. Reactome study revealed that both genes may interact more closely during the G1-G1/S phases of the cell's growth. As a result, inhibiting AURKA and CDC20 may hold promise as a cancer treatment.\n\nFurthermore, proteins involved in the cell cycle, such as CDC20 and AURKA we found that both cells expressed ACYL positively. ACLY genes encoded ATP-Citrate lyase, which converts citrate to acetyl-CoA and oxaloacetate (Zaidi, Swinnen, & Smans, 2012). The enzyme is required for lipogenesis and its expression was shown to be increased in colorectal cancer compared to normal mucosa. Additionally, ACLY has been implicated in medication resistance (Zhou et al., 2013). ACLY expression has been shown to decrease cytosolic citrate while increasing glycolysis. Additionally, it was observed that ACLY activates the oncogenic PI3K/MAPK kinases, leading to the Warburg effect. Additionally, Warburg may promote ACLY expression within cells via feedback regulation (Icard et al., 2020). ACLY activity generates acetyl-CoA, a precursor for fatty acid synthesis, and the Mevalonate pathway, which is required for the generation of substrates for protein prenylation, such as geranylgeranyl-pyrophosphate and farnesyl-pyrophosphate. These changes facilitate malignant transformation, invasion, and metastasis (Zaidi, Swinnen, & Smans, 2012). Additionally, by stabilizing CTNNB1, ACLY displayed greater migration and invasion. As a result, ACLY was proposed as a primary therapeutic target for both treatment and prevention (Khwairakpam et al., 2014). To summarize, employing primary ex-vivo models may help to understand how cancers grow in vitro and may be useful for predicting either the tumor's genetic profile or its application as precision medicine.\n\n\nConclusions\n\nUsing a mix of mechanical disaggregation and three-dimensional culture techniques, we developed preclinical ex vivo models. These techniques were convenient and straightforward to employ, allowing for the rapid production of preclinical ex vivo models in 3–7 days. In ex vivo models, the cells produced ACLY, AURKA, and CDC20, which may be linked with key genes required for cell proliferation.\n\n\nData availability\n\nOpen Science Framework: Colorectal Cancer on a Dish: Exploring the 3D-sphere culture of primary colorectal cancer cells from an Indonesian perspective https://doi.org/10.17605/OSF.IO/DPYWQ (Abdullah, 2021).\n\nThis project contains the following underlying data:\n\n‐ Amplification Plot_CRC001.jpg\n\n‐ Amplification Plot_CRC002.jpg\n\n‐ CRC 001.tif\n\n‐ CT results - human cancer pathway finder.xlsx\n\n‐ disagreggation result - CRC 001.tif\n\n‐ Figure 1. tif (CRC 001 morphology)\n\n‐ Figure 2. tif (CRC 002 morphology)\n\n‐ Figure 3. png (Protein-Protein Interaction/PPI interaction of co-expressed genes)\n\n‐ Melt Curve_CRC001.jpg\n\n‐ Melt Curve_CRC002.jpg\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank Dr. Nadhira Nizam and Dr. Asiyah Nurul Fadilah for their valuable contribution in gathering patients and samples resources.\n\n\nReferences\n\nAbdullah M: Colorectal Cancer on a Dish: Exploring the 3D-sphere culture of primary colorectal cancer cells from an Indonesian perspective [Internet]. 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PubMed Abstract \| Publisher Full Text\n\nKardia E, Frese M, Strive T, et al.: Isolation, culture and maintenance of rabbit intestinal organoids, and organoid-derived cell monolayers. bioRxiv:2020.07.15.205328. 2020. Publisher Full Text\n\nKhwairakpam A, Mayengbam S, Sailo B, et al.: ATP Citrate Lyase (ACLY): A Promising Target for Cancer Prevention and Treatment. Curr. Drug Targets. 2014; 16: 156–163. PubMed Abstract \| Publisher Full Text\n\nKidokoro T, Tanikawa C, Furukawa Y, et al.: CDC20, a potential cancer therapeutic target, is negatively regulated by p53. Oncogene. 2008; 27: 1562–1571. PubMed Abstract \| Publisher Full Text\n\nKoh HM, Jang BG, Hyun CL, et al.: Aurora Kinase A Is a Prognostic Marker in Colorectal Adenocarcinoma. J. Pathol. Transl. Med. 2017; 51: 32–39. PubMed Abstract \| Publisher Full Text\n\nKumar B, Singh S, Skvortsova I, et al.: Promising Targets in Anti-cancer Drug Development: Recent Updates. Curr. Med. Chem. 2017; 24: 4729–4752. PubMed Abstract \| Publisher Full Text\n\nKurashina Y, Imashiro C, Hirano M, et al.: Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves. Commun. Biol. 2019; 2: 393. PubMed Abstract \| Publisher Full Text\n\nMeijer TG, Naipal KA, Jager A, et al.: Ex vivo tumor culture systems for functional drug testing and therapy response prediction. Future science OA. 2017; 3: FSO190–FSO190. PubMed Abstract \| Publisher Full Text\n\nMirabelli P, Coppola L, Salvatore M: Cancer Cell Lines Are Useful Model Systems for Medical Research. Cancers. 2019; 11: 1098. PubMed Abstract \| Publisher Full Text\n\nMiserocchi G, Mercatali L, Liverani C, et al.: Management and potentialities of primary cancer cultures in preclinical and translational studies. J. Transl. Med. 2017; 15: 229. PubMed Abstract \| Publisher Full Text\n\nMondal G, Sengupta S, Panda CK, et al.: Overexpression of Cdc20 leads to impairment of the spindle assembly checkpoint and aneuploidization in oral cancer. Carcinogenesis. 2007; 28: 81–92. PubMed Abstract \| Publisher Full Text\n\nPerry WL 3rd, Weitzman A: The development of molecularly targeted anticancer therapies: an Eli Lilly and Company perspective. Clin. Adv. Hematol. Oncol. 2005; 3: 199–202. 237–238. PubMed Abstract\n\nPetit V, Massonnet G, Maciorowski Z, et al.: Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters. Lab. Investig. 2013; 93: 611–621. PubMed Abstract \| Publisher Full Text\n\nRiffle S, Hegde RS: Modeling tumor cell adaptations to hypoxia in multicellular tumor spheroids. J. Exp. Clin. Cancer Res. 2017; 36: 102–102. PubMed Abstract \| Publisher Full Text\n\nStrickaert A, Saiselet M, Dom G, et al.: Cancer heterogeneity is not compatible with one unique cancer cell metabolic map. Oncogene. 2017; 36: 2637–2642. PubMed Abstract \| Publisher Full Text\n\nTakahashi Y, Sheridan P, Niida A, et al.: The AURKA/TPX2 axis drives colon tumorigenesis cooperatively with MYC. Ann. Oncol. 2015; 26: 935–942. PubMed Abstract \| Publisher Full Text\n\nThomas RM, Van Dyke T, Merlino G, et al.: Concepts in Cancer Modeling: A Brief History. Cancer Res. 2016; 76: 5921–5925. PubMed Abstract \| Publisher Full Text\n\nWen J, Min X, Shen M, et al.: ACLY facilitates colon cancer cell metastasis by CTNNB1. J. Exp. Clin. Cancer Res. 2019; 38: 401. PubMed Abstract \| Publisher Full Text\n\nWu G, Haw R: Functional Interaction Network Construction and Analysis for Disease Discovery. Methods in Molecular Biology (Clifton, N.J.). 2017; 1558: 235–253. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "144777", "date": "15 Aug 2022", "name": "Estela Solanas", "expertise": [ "Reviewer Expertise tissue culture methods", "in vitro models", "gastrointestinal diseases", "liver regeneration" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article aims to develop a method for the detection of colorectal cancer using an in vitro model of 3D spheres formed of cells from patient biopsies. For this purpose, the authors disaggregate tumour samples from 2 patients and seed and culture them on Nunc-Sphera 96-well plate for 7 days. On the spheres formed, the profile of genes involved in human cancer and the pathways involved were studied by qPCR, while protein-protein interactions from positively expressed genes was analysed using the String-8 Platform. The results support the expression of CDC20, AURKA, and ACLY in these tumour models. These markers have been implicated in metastasis, CRC proliferation, and as a drug target ligand. They conclude that the presented model may allow CRC detection in developing countries.\n\nSome important aspects are not well explained in the text and are not clear. One of them is the aim of the study. The authors want to develop a CRC screening method to use in resource-poor countries, but the proposed method seems to be more costly and complicated than a pathological analysis of biopsies. Therefore, it would be more understandable that the aim would be to develop a model for personalised CRC screening and drug testing. It is also unclear at what time/s the different analyses are performed, whether at 7 or 14 days post culture, and the number of samples used in each patient.\nAs for the methodology used, the way in which the spheres are obtained is explained in the results section instead of in the methodology section, where it should be. In this regard, the exact time of culture is not detailed, nor when exactly the analyses are carried out (in some places it is said that they are cultured for 7 days and in others that they are analysed after 14 days of culture...).\n\nAs for the statistical analysis, it is not detailed or mentioned in the methodology section, although the existence of significant differences between samples is mentioned in the discussion. Therefore, it would be necessary to detail the type of analysis, number of samples per patient, etc., since if samples from each patient are analysed and compared, a specific statistical analysis is required.\n\nA very important methodological aspect to take into account is that the genetic profile of the patient's biopsies is not studied or not detailed, which is fundamental to know if the profile found in the spheres is the same as that of the original tumour from which they originate and therefore if the proposed model or method is valid. Also in this sense, not finding expression for housekeeping genes and not being able to normalise the expression of the rest of the genes does not allow us to compare the expression of the different samples and much less to compare the profiles of different patients, invalidating to a large extent the conclusions obtained. It would be necessary to study the genetic profile of the original tumours of the patients and see if it corresponds to that found in the spheres in culture. Nor is there a study of whether the genes found are characteristic of CRC or whether they are associated with one stage or another of the tumour?\nOn the other hand, the images of the spheres obtained from the different patients are taken with different lenses (10x and 20x), so visual comparison is not possible.\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": "8695", "date": "31 Aug 2022", "name": "Murdani Abdullah", "role": "Author Response", "response": "\"This article aims to develop a method for the detection of colorectal cancer using an in vitro model of 3D spheres formed of cells from patient biopsies. For this purpose, the authors disaggregate tumour samples from 2 patients and seed and culture them on Nunc-Sphera 96-well plate for 7 days. On the spheres formed, the profile of genes involved in human cancer and the pathways involved were studied by qPCR, while protein-protein interactions from positively expressed genes was analysed using the String-8 Platform. The results support the expression of CDC20, AURKA, and ACLY in these tumour models. These markers have been implicated in metastasis, CRC proliferation, and as a drug target ligand. They conclude that the presented model may allow CRC detection in developing countries. Some important aspects are not well explained in the text and are not clear. One of them is the aim of the study. The authors want to develop a CRC screening method to use in resource-poor countries, but the proposed method seems to be more costly and complicated than a pathological analysis of biopsies. Therefore, it would be more understandable that the aim would be to develop a model for personalised CRC screening and drug testing. It is also unclear at what time/s the different analyses are performed, whether at 7 or 14 days post culture, and the number of samples used in each patient.\" Indeed the aim in particular understanding genes that may related to development of CRC in Indonesia. for molecular analysis, cultures was harvested on day 7. In this paper, we want to share our experience for developing CRC in a dish using low plates and aggregation methods. \"As for the methodology used, the way in which the spheres are obtained is explained in the results section instead of in the methodology section, where it should be. In this regard, the exact time of culture is not detailed, nor when exactly the analyses are carried out (in some places it is said that they are cultured for 7 days and in others that they are analysed after 14 days of culture...).\" The exact times was approximately 7 days, and during the pandemic times and limited time to enter the lab. some was taken on day 14. \"As for the statistical analysis, it is not detailed or mentioned in the methodology section, although the existence of significant differences between samples is mentioned in the discussion. Therefore, it would be necessary to detail the type of analysis, number of samples per patient, etc., since if samples from each patient are analysed and compared, a specific statistical analysis is required.\" Statistical would be very good when we have sample size, however the study was conducted during pandemic times, while patients were very limited and enter the lab in a “normal schedule” would be very difficult. \"A very important methodological aspect to take into account is that the genetic profile of the patient's biopsies is not studied or not detailed, which is fundamental to know if the profile found in the spheres is the same as that of the original tumour from which they originate and therefore if the proposed model or method is valid. Also in this sense, not finding expression for housekeeping genes and not being able to normalise the expression of the rest of the genes does not allow us to compare the expression of the different samples and much less to compare the profiles of different patients, invalidating to a large extent the conclusions obtained. It would be necessary to study the genetic profile of the original tumours of the patients and see if it corresponds to that found in the spheres in culture. Nor is there a study of whether the genes found are characteristic of CRC or whether they are associated with one stage or another of the tumour?\" In a future study, correspond genetics or markers would be very interesting. however in this study we want to simply observed what genes are expressed during the growth of these culture. and not goes to more detailed comparative questions such as genetic alterations and so on. \"On the other hand, the images of the spheres obtained from the different patients are taken with different lenses (10x and 20x), so visual comparison is not possible.\" Yes, indeed. Figure 1 gave presentations of heterogeneity of “tumor core” while figure 2 just shown morphology. The main idea was because in figure 2 the tumor core are difficult to observed or specifically may not form heterogenity morphology as it shown in Figure 1." } ] }, { "id": "238092", "date": "07 Feb 2024", "name": "Triyanta Yuli Pramana", "expertise": [ "Reviewer Expertise antioxidant 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 article is about Colorectal cancer in Indonesia and developing a CRC detection method for Indonesia and other low-middle income nations that integrates in vitro 3D culture, molecular analysis, and in silico analysis.\nAre sufficient details of methods and analysis provided to allow replication by others?. Partly There is statement \"The PPI was enriched one time by clicking “more” and represented as images.\", difficult to understand. It's need more explanation about \"by clicking more\", so other researcher can perform similar technique.\nAre all the source data underlying the result available to ensure full reproducibility.. Partly. even 2 specimen are sound reproducible, but the number of specimen so small. it need more specimen. There is no data about these technique with Indonesia, beside it's performed in Indonesia, so the conclusion that a combination of 3D culture, gene profiling, and in silico analysis is feasible to detect CRC for Indonesia and other low- and middle-income countries, was not supported by data\nAre the conclusions drawn adequately supported by the results?. Partly The abstract conclusion say that silico analysis is feasible to detect CRC for Indonesia and other low- and middle-income countries. In the article there was no discussion and conclusion about Indonesia, besides these research was performed in Jakarta (Indonesia)\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": [] } ]	1	https://f1000research.com/articles/11-182
https://f1000research.com/articles/11-181/v1	14 Feb 22	{ "type": "Method Article", "title": "Vessel masking and Hough transform for optic disc localisation from retinal images", "authors": [ "Aziah Ali", "Wan Mimi Diyana Wan Zaki", "Aini Hussain", "Noramiza Hashim", "Wan Noorshahida Mohd Isa", "Wan Mimi Diyana Wan Zaki", "Aini Hussain", "Noramiza Hashim", "Wan Noorshahida Mohd Isa" ], "abstract": "Background: Retinal images can be considered as one of the reliable indicators for symptoms of many ocular diseases such as diabetic retinopathy, macular degeneration and glaucoma. By analysing and tracking changes of important structures on a retinal image, symptoms of ocular diseases can be detected in a timely manner which helps physicians plan early treatment for better disease control. One of the important landmarks on a retinal image is the optic disc (OD), which must be localised to estimate retinal vessel parameters such as vessel width and tortuosity. This paper proposes a method for automatic OD localisation from a retinal image. Methods: A retinal image is first pre-processed and thresholded to produce a binary image that highlights most retinal vessels on the image. Next, a discrete cosine transform-based smoothing method is employed to replace the detected vessel pixel values on the pre-processed image with values closer to the surrounding neighbour pixel values, effectively masking most vessels on the image. Hough transform is then applied to the vessel-masked image to detect the circle representing the OD on the image, producing the estimated location of the OD center and its estimated diameter. Results: Applying the proposed method to three different public databases, namely Digital Retinal Images for Vessel Extraction (DRIVE), High-Resolution Fundus (HRF) and Methods to Evaluate Segmentation and Indexing Techniques in the field of Retinal Ophthalmology (MESSIDOR) resulted in an overall detection rate of 99.53%. Conclusions: The achieved performance by the proposed method is superior to many published methods of OD localization, with a processing time of less than one second for each image. While this has only been validated on one type of retinal images, future investigations may include validation on other types such as angiograms or scanning laser ophthalmoscopy.", "keywords": [ "Optic disc localisation", "fundus image", "vessel masking", "Hough transform" ], "content": "Introduction\n\nRetinal image analysis can be very helpful in providing insights into patients’ ocular health. By analysing the retinal image, ophthalmologists can detect various symptoms of ocular diseases, which may help to ensure timely treatment of the diseases, thus ultimately decreasing the risk of patients going totally blind. Most hospitals are now equipped with modern fundus cameras that image the patient’s fundus to produce a retinal image. Figure 1 shows a sample of a fundus image. Nerves from the retina converge to form a round or oval optic disc (OD) that sends a focused image onto the retina, in the form of electrical impulses to the part of the brain responsible for visual function. The central part of the retina, known as the macula, is responsible for an important part of the central vision system, while the fovea is the point in the middle of the macula.\n\nWith routine retinal screening in place, a huge number of fundus images will need to be analysed daily. This scenario has resulted in a lot of research being conducted on the automatic analysis of fundus images to assist ophthalmologists in efficiently and accurately performing retinal diagnoses.1,2 These studies aim to extract important parameters from a fundus image, mostly related to the important landmarks, including the OD, retinal blood vessels, fovea, macula, and any associated anomalies.\n\nA topic of interest regarding fundus image analysis is the automatic localisation of the OD from a fundus image. By detecting the OD, parameters such as its position and radius could be used to estimate other parameters such as vessel width or tortuosity. Normally, when measuring for these parameters from a fundus image, to be considered for parameter calculation the vessels are to be of certain distance close to the OD.3 OD detection would also allow for identification of the eye side from which the image is taken, whether right eye or left eye.\n\nA number of studies have been dedicated to automatically detecting the OD on the fundus image,4–8 while others have also attempted at providing a more accurate boundary of the detected OD.9–12 The methods used include circular transformation,11 directional local contrast,13 probability models,6 automatic thresholding3 and deep learning.12,14\n\nThresholding works in locating the OD in fundus images with high-intensity differences between the OD region and other parts of the image.15–18 When dealing with images containing an OD with low contrast against the retinal background or images with pathologies, the thresholding method may fail to detect the OD.19 A set of points is used to describe the OD boundary by minimising the energy function in active contour-based methods for OD detection.8,20–22 While this method may work well, its performance is very much dependent on the initial seed points for the contour model. There is also the risk of being trapped in a local maximum when searching for the OD boundary, especially with images containing pathologies. Extensive review of existing OD segmentation methods can be found in the review literature.19,23\n\nOD localisation focuses more on locating the position of OD center on the fundus image, which is different from the focus of the OD segmentation procedure. In OD segmentation, the general aim is to identify every pixel that belongs to the OD on the fundus. In most applications, OD localisation precedes the OD segmentation step; hence it is important to have an accurate estimate of the OD center through OD localisation to ensure a successful OD segmentation procedure. Since the OD is usually a bright disc-shaped area on the fundus image, some researchers have investigated the use of the Hough transform technique to detect the shape and thus estimate the center of the OD.3,24–27 Many researchers employ methods to remove vessel structures from the fundus image, or use vessel masking, to further highlight the OD structure, such as inpainting28 and median filtering.24 Combining Hough transform with vessel masking can be a potential method to efficiently localise the OD in a fundus image, instead of using the methods separately.24–28\n\n\nMethods\n\nA method inspired by combining existing efficient OD localisation methods, namely thresholding, vessel masking and Hough transform, is proposed to localise the OD centre's position from a fundus image.\n\nThe proposed OD localisation method takes a color fundus image as the input. Firstly, the green channel image is extracted from the color image as part of pre-processing. Next, the green channel image is padded around the original region of interest (ROI - the circular non-black area), with additional pixels matching the pixel values along the border. This pre-processing step is similar to Soares’ proposed method29 for retinal vessel segmentation, except that the number of iterations for ROI padding is increased to 50 instead of 20. This step helps to minimise the contrast between the ROI and background further so it would not be falsely detected as the OD centre in the following step. The pre-processed image is then resized to a standardised smaller size for faster computation and is converted to a binary image using a global thresholding method, called Otsu’s method. The binary image will highlight most vessel structures in the pre-processed fundus image in white pixels, while the retinal background is in black pixels. This method is implemented using the Matlab software, which can potentially be translated into SCILAB as an open-source alternative.\n\nNext, using the vessel pixel information from the binary image, a discrete cosine transform-based smoothing method is employed on the pre-processed image to replace all the vessel pixel values with values closer to the surrounding neighbours. This vessel masking step will effectively remove most of the vessel structures from the image, resulting in a vessel-masked image. The Hough transform is then applied to detect the circle representing the OD on the image. Once the circle has been detected, the OD centre and the radius can then be estimated to be used in the estimation of important retinal parameters such as cup-to-disc ratio, tortuosity and calibre of the retinal vessels.\n\nFigure 2 depicts all steps involved in OD localisation from a fundus image and their corresponding sample output images.\n\nFor validation, it is not necessary for the fundus images to have ground truth vessel segmentation images. In a number of previous studies on OD localisation, a database called Methods to Evaluate Segmentation and Indexing Techniques in the field of Retinal Ophthalmology (MESSIDOR) is used for validation.9,11,24,30,31 The MESSIDOR database32 consists of 1200 fundus images captured using a Topcon TRC NW6 non-mydriatic fundus camera with 45 degrees of field of viev (FOV). In this study, the OD’s centre position and radius are estimated on all 40 images from Digital Retinal Images for Vessel Extraction (DRIVE),33 45 images from High-Resolution Fundus (HRF),34 and 1200 images from MESSIDOR, which are all publicly available fundus image databases. The images from another popular benchmark fundus image database, the STructured Analysis of the Retina (STARE) database, are excluded in this evaluation since most of its images do not contain OD. Even for those with the OD in the ROI, the OD is only partially visible.\n\n\nResults & discussion\n\nTable 1 shows sample output images for the main steps in the proposed OD localisation method for the DRIVE, HRF and HUKM databases. The OD-localised image output includes a “+” sign to indicate the estimated OD centre and the green circle denotes the estimated OD radius. Figure 3 shows zoomed-in images of the OD localisation output from HRF images. It can be seen that the proposed method managed to accurately detect the centre and the radius of the OD, regardless of whether the fundus image contains a clean (normal) or noisy (with pathologies) retinal background.\n\nFollowing the previous researchers’ method of assessing the OD localisation performance, a method is considered to have successful OD localisation when the estimated location of OD center is within the circumference of the OD itself.31 The proposed OD localisation method achieves a 100% correct detection rate for all images in DRIVE and HRF. For the larger MESSIDOR database, only six images out of 1200 images result in either wrong detection or non-detection of the OD, hence 99.5% successful rate. These results are summarised in Table 2 below.\n\nTable 3 shows the performance comparison of the proposed method against published methods in the literature. The proposed method outperforms all considered methods except for Yu’s method that achieved 99.67% detection rate. On average, this translates to a 99.53% detection rate for all the validated databases. The processing time for OD localisation is less than one second for every image tested, regardless of the original image resolution. Shorter processing time is achieved because the proposed method employs image resizing, however this does not compromise the detection rate. This method is efficient and accurate for practical application of OD localisation in clinical settings.\n\n\nConclusion\n\nIn this paper, we have proposed an efficient method for localising the OD in a fundus image. The method involves the use of vessel masking to remove vessel structures from the image and Hough transform to locate the circular object on the vessel-masked image, which is the OD. The output will be in the form of the coordinates of the OD center together with the estimated radius of the OD, which can also be visualised on the fundus image. Validation of the proposed method on three different public databases, namely DRIVE, HRF and MESSIDOR resulted in an overall detection rate of 99.53%. The achieved performance is superior to many published methods available, with a much-reduced processing time of less than one second for each image. The proposed method has only been validated on one type of retinal image, which is a fundus image produced by a fundus camera. In the future, retinal images using other imaging modalities such as angiogram or scanning laser ophthalmoscopy can further validate the proposed optic disc localisation. Another interesting direction for future research is accurate segmentation of the OD boundary for more accurate parameter estimation. The output of the method may prove to be useful for diagnosing ocular diseases, which relate to parameters such as cup-to-disc ratio and vessel width parameters. Automating the step for OD localisation can help develop a fully automated computer-assisted retinal diagnosis system in the future.\n\n\nData availability\n\nThe DRIVE database can be accessed at https://drive.grand-challenge.org/, HRF at https://www5.cs.fau.de/research/data/fundus-images/ and MESSIDOR at https://www.adcis.net/en/third-party/messidor/.", "appendix": "Acknowledgments\n\nWe would like to thank our collaborators from the Department of Ophthalmology, Universiti Kebangsaan Malaysia Medical Center, especially Dr Wan Haslina and her team for their valuable inputs for this study.\n\n\nReferences\n\nChalakkal RJ, Abdulla WH, Hong SC: Fundus retinal image analyses for screening and diagnosing diabetic retinopathy, macular edema, and glaucoma disorders. Diabetes and Fundus OCT. Elsevier; 2020; pp. 59–111. Publisher Full Text\n\nMittal K, Rajam VMA: Computerised retinal image analysis - a survey. Multimed. Tools Appl. Aug. 2020; 79(31–32):22389–22421. 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Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics). 2017.\n\nZhang D, Zhao Y: Novel accurate and fast optic disc detection in retinal images with vessel distribution and directional characteristics. IEEE J. Biomed. Heal. Informatics. 2016.\n\nYu S, Xiao D, Frost S, et al.: Robust optic disc and cup segmentation with deep learning for glaucoma detection. Comput. Med. Imaging Graph. Jun. 2019; 74: 61–71. PubMed Abstract \| Publisher Full Text\n\nCheng J, et al.: Automatic optic disc segmentation with peripapillary atrophy elimination. Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS. 2011; pp. 6224–6227.\n\nIssac A, Sengar N, Singh A, et al.: Automated computer vision method for optic disc detection from non-uniform illuminated digital fundus images. 2nd International Conference on Communication, Control and Intelligent Systems, CCIS 2016. 2016.\n\nJoshi GD, Sivaswamy J, Karan K, et al.: Optic disk and cup boundary detection using regional information. 2010 7th IEEE International Symposium on Biomedical Imaging: From Nano to Macro, ISBI 2010 - Proceedings. 2010; pp. 948–951.\n\nNoor NM, Khalid NEA, Ariff NM: Optic cup and disc color channel multi-thresholding segmentation. Proceedings - 2013 IEEE International Conference on Control System, Computing and Engineering, ICCSCE 2013. 2013; pp. 530–534.\n\nThakur N, Juneja M: Survey on segmentation and classification approaches of optic cup and optic disc for diagnosis of glaucoma. Biomedical Signal Processing and Control. 01-Apr-2018; 42: 162–189. Elsevier. Publisher Full Text\n\nElbalaoui A, Fakir M, Ouadid Y, et al.: Boundary segmentation of optic disc in fundus images. Proceedings - 2017 14th International Conference on Computer Graphics, Imaging and Visualization, CGiV 2017. 2018.\n\nGanesan P, Sathish BS: Automatic detection of optic disc and blood vessel in retinal images using morphological operations and ipachi model. Res. J. Pharm. Technol. 2017; 10: 2602. Publisher Full Text\n\nGiraddi S, Pujari J, Hiremath PS: Optic disc detection using geometric properties and GVFsnake. Proceedings - 1st International Conference on Intelligent Systems and Information Management, ICISIM 2017. 2017.\n\nMary MCVS, Rajsingh EB, Naik GR: Retinal Fundus Image Analysis for Diagnosis of Glaucoma: A Comprehensive Survey. IEEE Access, Institute of Electrical and Electronics Engineers Inc. ;2016; vol. 4: pp. 4327–4354. Publisher Full Text\n\nAbdullah M, Fraz MM, Barman SA: Localisation and segmentation of optic disc in retinal images using Circular Hough transform and Grow Cut algorithm. PeerJ. 2016; 4: e2003. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChalakkal RJ, Abdulla WH, Thulaseedharan SS: Automatic detection and segmentation of optic disc and fovea in retinal images. IET Image Process. 2018; 12: 2100–2110. Publisher Full Text\n\nFigueiredo IN, Kumar S: Automatic optic disc detection in retinal fundus images based on geometric features. Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics). 2014.\n\nSekhar S, Al-Nuaimy W, Nandi AK: Automated localisation of retinal optic disk using hough transform. 2008 50th IEEE International Symposium on Biomedical Imaging: From Nano to Macro, Proceedings, ISBI. 2008; pp. 1577–1580.\n\nAlmazroa A, Sun W, Alodhayb S, et al.: Optic disc segmentation: level set methods and blood vessels inpainting. Medical Imaging 2017: Imaging Informatics for Healthcare, Research, and Applications. 2017.\n\nSoares JVB, Leandro JJG, Cesar RM Jr, et al.: Retinal vessel segmentation using the 2-D Gabor wavelet and supervised classification. Med. Imaging, IEEE Trans. 2006; 25(9): 1214–1222. Publisher Full Text\n\nSalih ND, Saleh MD, Eswaran C, et al.: Fast optic disc segmentation using FFT-based template-matching and region-growing techniques. Comput. Methods Biomech. Biomed. Eng. Imaging Vis. 2018; 6: 101–112. Publisher Full Text\n\nYu T, Ma Y, Li W: Automatic localisation and segmentation of optic disc in fundus image using morphology and level set. International Symposium on Medical Information and Communication Technology, ISMICT. 2015; vol. 2015-May: pp. 195–199.\n\nDecencière E, et al.: Feedback on a publicly distributed image database: The Messidor database. Image Anal. Stereol. Aug. 2014; 33(3): 231–234.Publisher Full Text\n\nStaal J, Abràmoff MD, Niemeijer M, et al.: Ridge-based vessel segmentation in color images of the retina. IEEE Trans. Med. Imaging. 2004; 23(4): 501–509. PubMed Abstract \| Publisher Full Text\n\nBudai A, Bock R, Maier A, et al.: Robust Vessel Segmentation in Fundus Images. Int. J. Biomed. Imaging. Dec. 2013; 2013: 1–11. Publisher Full Text" }	[ { "id": "123841", "date": "16 Nov 2023", "name": "Khan Bahadar Khan", "expertise": [ "Reviewer Expertise Retinal Vessel segmentation", "Biomedical Image Processing" ], "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\nFollowing are some suggestions to improve the current work:\nThis paper is an application-based technical paper, which adopts a well-known method. However, it is short of novelty.\n\nIn section 1 (Introduction), it’s better to clarify the whole structure of the paper, which can be easier for the reader to understand what scenarios have been investigated or examined and what’s the main contribution this paper makes should be highlighted in bullet form for the ease of the readers. For example, this paper is organized as follows: section 2 presents what and section 3 presents what… It’s suggested to give a brief overview of the entire paper in the introduction.\n\nThe authors are advised to refer to more datasets or some newer fundus datasets. The authors must consider the clinical images and the results must be evaluated by the physicians. The following latest databases are available (Samiksha et al. 2020; Muhammed et al. 2006, and Ce et al. 2021).1 2 3\n\nIn the related work, the authors have described several previous methods for the problem. They could explain how their work differs from or improves existing methods.\n\nIt would be better to compare the obtained results with existing state-of-the-art techniques for better judgment of the proposed approach.\n\nPlease add the discussion section separately. The discussion section is always important while making a comparison of the findings with previous studies. Therefore, it is needed to compare the results of the study with existing literature. Are the findings being in line with the literature? If it is not inline then why the findings are apposite to the literature? This needs to be addressed.\n\nThere are some minor comments about the paper. There are still some errors and typos in the paper. Your submission requires extensive editing for English grammar and usage. Please have your manuscript copy-edited by a professional copy-editing service. The format of all references should be uniform and all references should be correctly listed.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? No\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\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? Partly", "responses": [] }, { "id": "150516", "date": "16 Nov 2023", "name": "Francisco Maria Calisto", "expertise": [ "Reviewer Expertise Human-Computer Interaction", "Health Informatics", "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\nIn this manuscript, the authors are proposing an efficient method for localizing the optic disc in a fundus image. Their method involves the use of vessel masking to remove vessel structures from the image. Additionally, the authors applied the Hough transform method to locate the circular object on the vessel-masked image, which is the optic disc. The authors validated properly the proposed method on three different public databases, namely DRIVE, HRF and MESSIDOR resulted in an overall detection rate of 99.53%. In the end, they demonstrate that the achieved performance is superior to many published methods available, with a much-reduced processing time of less than one second for each image. Another interesting direction that the authors were discussing, is the potential to promote an accurate segmentation of the optic disc boundary for more accurate parameter estimation. This represents an exciting achievement. Not only, the authors are demonstrating good results with the proposed method, but also are showing promising directions of the work.\nThe submitted manuscript, under revision, is well and clearly structured. The introduction of the work gives a short brief of the domain problem, while the background describes that both domain problem and scientific community can benefit from the procedure itself obtained by the provided methods [1, 5, 8]. Previous literature gives an overview of the related work to the resolution enhancement and its influence on the technique performance [3, 7, 9].\nProposed methods are giving enough details to the concern resolution, while presenting several methods, including the Hough transform. It also describes the approaches used for the analysis. However, it could also be interesting to discuss how clinical adoption is working with these new set of methods [2], as well as how can the authors apply these methods to other clinical domains [4, 6].\nAs a main opinion, this manuscript gives a good presentation of the contribution impact on the domain tasks. Authors are presenting their sampling technique, stating its novelty. The mentioned existing methods are well referenced in the manuscript.\nThe proposed method is clearly described, and the manuscript is well organized. The method description should be clear for readers with technical background. All the detailed in depth information are provided and described with support of results. Validation procedure of the proposed methods are satisfactory.\nFinally, it would be interesting to see similar analysis for the proposed methods. In summary, the manuscript shows an exciting work and can be recommended for publication.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-181
https://f1000research.com/articles/11-176/v1	14 Feb 22	{ "type": "Case Report", "title": "Case Report: Ovarian metastasis revealing primary gall bladder carcinoma", "authors": [ "Hajer Bettaieb", "Nesrine Souayeh", "Mehdi Ben Nar", "Sana Mezghani", "Fatma Khanchel", "Wael Mbarki", "Safa Laifi", "Imen Helal", "Hedhili Oueslati", "Najeh Hsayaoui", "Chaouki Mbarki", "Hajer Bettaieb", "Nesrine Souayeh", "Mehdi Ben Nar", "Sana Mezghani", "Fatma Khanchel", "Wael Mbarki", "Safa Laifi", "Imen Helal", "Hedhili Oueslati", "Najeh Hsayaoui" ], "abstract": "Ovaries can be an elective metastatic site for many cancers mostly in gastrointestinal tract, breast and endometrial cancers. Gallbladder and bile ducts cancers are rare, accounting for few ovarian metastatic tumors. We report a case of bilateral metastatic ovarian of gallbladder carcinoma in a 44 years old woman. Patient had pelvic pain, anorexia, weight loss and decreased visual acuity. Imagery showed bilateral ovarian tumor and polyp in the gallbladder. Furthermore, there were metastasis to the liver, lungs and choroid plexus. Liver biopsy confirmed the metastatic origin from gallbladder cancer. Patient underwent surgery. She had total hysterectomy with bilateral oophorectomy, omentectomy and cholecystectomy. She passed away six months later, after refusing additional adjuvant therapy.", "keywords": [ "gallbladder carcinoma", "ovarian metastasis", "radiology", "pathology" ], "content": "Introduction\n\nOvaries can be an elective metastatic site for many cancers mostly in gastrointestinal tract, breast and endometrial cancers. Krukenberg tumor also known as signet ring adenocarcinoma, mostly from gastric origin, is the most described.1 Only a few cases of ovarian gallbladder cancer metastasis are reported in the literature.1,2 The chance of finding ovarian spread among patients with gallbladder carcinoma are up to six percent.1,2 Here we describe a case of bilateral ovarian metastasis of gallbladder carcinoma in a 44-year-old woman, suffering from pelvic pain, anorexia, weight loss and decreased visual acuity. She declined adjuvant therapy and passed away six months after surgery.\n\n\nCase report\n\nA 44-year-old Arabic woman (gravida 4, para 3) was admitted to our department of gynecology suffering from chronic pelvic pain associated with weight loss and anorexia during the last three months and had been experiencing reduced visual acuity since a week. She had no relevant medical history and did not undergo any surgical procedure. No family history of inherited gynecologic cancer was found. A physical examination found bilateral pelvic masses. An ultrasound exam showed bilateral and complex ovarian cysts with septa. Serum tumor markers (CA-125, CEA and CA19-9) were normal. A CT scan and pelvic abdominal MRI were performed. They showed bilateral ovarian masses with large cysts and enhanced septa. The tumors were isodense on the CT scan and hyper intense on T2 MRI sequences (Figure 1). We additionally found several necrotic metastatic lesions in the liver (segments I, IV, VI and VII) associated to a polyp of the gallbladder (Figure 2). Furthermore, there were metastatic microndular miliary in lungs, left pulmonary arterial embolism and bilateral choroid metastasis (Figure 3). Liver biopsy showed abnormal cell proliferation arranged in glandular structures, backed against each other. Tumor cells were cytokeratin 7 and 19 positive, cytokeratin 20 and hepatocyte antigen negative, allowing the diagnosis of a moderately differentiated adenocarcinoma arising from the gallbladder.\n\nMultidisciplinary consultation meeting decided to perform primary staging surgery (total abdominal hysterectomy, bilateral adnexectomy, omentectomy, peritoneal biopsy, cholecystectomy and partial hepatectomy). Per-operative exploration showed suspicious bilateral mi part solid mi part cyst ovarian tumors, with a focally roughened surface. Uterus was normal in shape and size. Gallbladder had thick walls. Pathologic examination of gallbladder showed moderately differentiated adenocarcinoma. However, it showed benign ovarian serous cystadenomas with deposits of metastatic adenocarcinoma in the parenchyma (Figure 4). There were no malignancy signs in uterus, omentum and peritoneal biopsies. Patient did not present any post-operative complications. After surgery, she was referred to the oncologists for adjuvant therapy. Unfortunately, the patient refused any further treatment and died six months later.\n\n\nDiscussion\n\nGallbladder carcinoma is found in 1% of patients undergoing cholecystectomy.2 Adenocarcinoma counts for 70 to 90% of gallbladder cancers.2 However, only a small percentage of patients with carcinoma of the gallbladder will ever have metastasis to the ovary.1,2 When ovarian tumors happen to be discovered prior to gallbladder carcinoma, it often leads to misinterpretation.\n\nWhen ovarian tumors are the first manifestation of the disease, gallbladder carcinoma spread to the ovaries can be misinterpreted for primary ovarian carcinoma.2,3 In fact, cases of gallbladder carcinoma do not infrequently present with hepatobiliary symptoms. However, when there is metastasis to the ovary, the biliary symptoms may be masked by ascites or local symptoms correlated to the ovarian mass.3,4 In a few cases, the primary gallbladder carcinoma was unsuspected and was discovered incidentally by surgical investigation during laparotomy for ovarian resection.3,4 In our case, the gallbladder carcinoma was suspected by the biopsy of the liver.\n\nAlthough there are a lot of data in the literature on ovarian metastases, the case of ovarian metastasis from gallbladder cancer remains little discussed.3-5 Accurate preoperative diagnosis of these advanced primary gallbladder carcinomas with ovarian metastases is less than 30%.5\n\nMany criteria have been reported to differentiate ovarian metastases from primary ovarian cancer.1,5 Bilaterality, surface implants, multinodularity, infiltrative pattern, growth in the ovarian hilum, mucin without epithelial cells on the tumor surface and presence of signet ring cells are the most common features suggesting ovarian metastasis.1,5 These features may be absent when the ovarian metastasis simulates a benign cyst.1 In our case report, the tumor was a mixture of solid and cystic areas. Brown et al.6 found in their revue of literature that most metastatic neoplasm to the ovary are predominantly solid or a mixture of solid and cystic areas and tended to be bilateral more often than primary neoplasm.\n\nAs for imaging criteria, Kim et al.7 found out, through a review of 85 patients’ CT imaging, that metastatic ovarian neoplasm should be suspected on CT examination when the tumor is solid and contained well defined intra-luminal cystic lesions.\n\nOn macroscopic examination, Lee and Young8 concluded that bilateral tumors with surface implants are in favor of metastatic neoplasm to the ovary.\n\nThis case’s originality lies in the fact that we spotted the primary tumor though it is rare and uncommon. The prognosis was poor from the beginning, worsened by the patient’s decision refusing any adjuvant therapy.\n\n\nConclusion\n\nOvarian metastasis can be difficult to distinguish from primary ovarian carcinoma especially when the primary gastrointestinal tract tumor is not yet diagnosed. There are no specific macroscopic or microscopic features that can differentiate ovarian metastases from a primary malignancy. Nevertheless, possibility of metastatic origin should be considered whenever we deal with a bilateral and/or mixed ovarian mass.\n\n\nConsent\n\nWe obtained informed consent from the patient’s family to publish the details of the case report.", "appendix": "References\n\nKumar Y, Chahal A, Garg M, et al.: Occult gallbladder carcinoma presenting as a primary ovarian tumor in two women: two case reports and a review of the literature. J Med Case Rep. 2010; 4: 202–209. PubMed Abstract \| Publisher Full Text\n\nJarvi K, Kelty CJ, Thomas WE, et al.: Bilateral ovarian metastases from carcinoma of the gallbladder. Gynecol Oncol. 2006; 103(1): 361–362. PubMed Abstract \| Publisher Full Text\n\nAyhan A, Guney I, Saygan-Karamursel B, et al.: Metastasis of primary biliary and gallbladder carcinomas. Eur J Gynaecol Oncol. 2001; 22: 377–378. PubMed Abstract\n\nJain V, Gupta K, Kudva R, et al.: A case of ovarian metastasis of gall bladder carcinoma simulating primary ovarian neoplasm: diagnostic pitfalls and review of literature. Int J Gynecol Cancer. 2006; 16: 319–321. Publisher Full Text\n\nKhunamornpong S, Lerwill MF, Siriaunkgul S, et al.: Carcinoma of extrahepatic bile ducts and gallbladder metastatic to ovary. A report of 16 cases. Int J Gynecol Pathol. 2008; 27: 366–379. Publisher Full Text\n\nBrown DL, Zou KH, Tempany CM, et al.: Primary versus secondary ovarian malignancy: imaging findings of adnexal masses in the radiology diagnostic oncology group study. Radiology. 2001; 219: 213–218. PubMed Abstract \| Publisher Full Text\n\nKim SH, Kim WH, Park KJ, et al.: CT and MR findings of Krukenberg tumours: comparison with primary ovarian tumors. J Comput Assist Tomogr. 1996; 20: 393–398. PubMed Abstract \| Publisher Full Text\n\nLee KR, Young RH: The distinction between primary and metastatic mucinous carcinomas of the ovary: gross and histologic findings in 50 cases. Am J Surg Pathol. 2003; 27: 281–292. Publisher Full Text" }	[ { "id": "203244", "date": "29 Sep 2023", "name": "Raquel Almeida", "expertise": [ "Reviewer Expertise Cancer researcher" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBettaieb et al report a rare case of gall bladder adenocarcinoma with ovarian metastases. The main interest of this case, is that ovarian metastases are a rare event and can be confounded with ovarian tumors. in this case, ovarian metastases were the first sign of tumor to be identified, but due to some peculiarities namely the bilaterally occurrence of ovarian tumors led to the suspicion that they could be metastases. Based on a well conducted inspection, with imaging and biopsies, the gall bladder primary tumor was identified which allowed to offer the patience the best treatment. This is thus informative for the clinicians and raises attention to the relevant features that need to be considered in order to distinguish primary ovarian tumors from ovarian metastases.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-176
https://f1000research.com/articles/10-1307/v1	22 Dec 21	{ "type": "Research Article", "title": "Childhood malnutrition and hypo mineralized molar defects: a cross sectional study", "authors": [ "Hoda Atef Abdelsattar Ibrahim", "Rania Abdallah Nasr", "Ahmed Adel Salama", "Aya Ahmed Amin", "Rania Abdallah Nasr", "Ahmed Adel Salama", "Aya Ahmed Amin" ], "abstract": "Background: Malnutrition is well-known to yield high morbidities and mortalities and considering its consequence on the oral cavity, malnutrition is shown to have pre-eruptive and post-eruptive outcomes. The objective was to assess the prevalence of hypo-mineralized second primary molar (HSPM), molar–incisor hypo-mineralization (MIH) and dental caries in malnourished children as well as addressing the relation between types of malnutrition of the children and their dental morbidities represented in HSPM, MIH and dental caries. Methods: This is a cross sectional analytical study. Malnourished children aged 5-10 years and presented to the Outpatient Clinic of Pediatric Dentistry Department, Faculty of Dentistry, Cairo University were examined for HSPM. MIH using European Academy of Pediatric Dentistry criteria and dental Caries using def/ DMF indices. Results: A consecutive sample (a long six months) of 54 malnourished children were enrolled in the study. Dental caries was a greater dental morbidity in the overweight and obese group. Besides, stunting was a greater risk in HSPM and MIH. There was an association between HSPM and MIH in a considerable percentage of the study group. Conclusions: Malnutrition is a risk factor for dental abnormalities. HSPM could expect the presence of MIH.", "keywords": [ "HSPM", "MIH", "dental caries", "Malnutrition", "children" ], "content": "Introduction\n\nMalnutrition is an essential determinant of morbidity and mortality in young children. It is linked with 45 percent of all deaths in children below five years of age.1,2 Malnutrition in children can include being underweight, being overweight, obesity, stunted growth and wasting.3 Malnutrition and its subtypes have been linked to dental comorbidities. For example, hypo mineralization and dental caries have been linked to stunted growth and obesity respectively. In addition, co-existence between these dental disorders can occur.4-6 Hypo mineralization represents the clinical existence of developmental defects which can be seen as discoloration, opacities or as a mixture of change in appearance and loss of enamel substance.7 Presently, there has been increasing attention regarding the fact that hypo mineralization can be a sign of interruption in a child’s growth as a result of early childhood illnesses.8,9\n\nCurrently, it has been addressed that a hypo-mineralized second primary molar (HSPM) could be a clinically important predictor for molar–incisor hypo-mineralization (MIH).10\n\nDental caries is a multifactorial disease. Factors affecting the onset of this disease could include diet composition, oral hygiene and frequency, socioeconomic status, bacterial load, salivary immunoglobulins and fluoride intake.11 It is determined as the single most common chronic disease during childhood.12\n\nDental comorbidities can coexist together. The increased caries risk concomitant with hypo-mineralization results is a considerable dental morbidity that is often culminating in subsequent orthodontic consequences.13\n\nThe study is aiming at assessing the dental abnormalities (HSPM, MIH, dental caries and co-occurrence of HSBM and MIH) in the malnourished children. The primary objective of the study is to detect the prevalence of HSPM, MIH, dental caries and co-occurrence of HSBM and MIH in malnourished children. Secondary objectives involve evaluating the association between HSPM, MIH, and dental caries with co-occurrence of HSBM and MIH, they also include assessing the association between the socioeconomic level, the type of malnutrition and dental abnormalities.\n\n\nMethods\n\nObservational cross sectional analytical study.\n\nOutpatient Clinic of Pediatric Dentistry Department, Faculty of Dentistry, Cairo University. The patient enrollment was from 1st of April to end of September, 2021.\n\nEligibility criteria\n\nInclusion criteria: 1. Children aged from 5 to 10 years. 2. Both genders. 3. All children who were following in the dental clinic and were observed to be malnourished along the duration of the study (a long six months duration). 4. Children whose parents or caregivers agreed to be enrolled in the study.\n\nExclusion criteria: 1. Children with extracted primary second molars and permanent incisors and molars. 2. Children with history of dental trauma. 3. Children with orthodontic bands or dental appliances. 4. Children whose parents or caregivers didn’t agree to be enrolled in the study.\n\n\n\n1. A consecutive sample of malnourished children aged from 5 to 10 years attending the outpatient clinic in Pediatric Dentistry Department, Faculty of Dentistry, Cairo University were included in this study according to eligibility criteria.\n\nDefinitions of malnutrition in the study:\n\nA. Underweight: when the weight for age is less than the mean by 2 standard deviations (SD) of the World Health Organization (WHO) Child Standards for growth or less than 3rd centile for age.\n\nB. Stunting: when the height for age is less than the mean by 2 standard deviations of the WHO Child Standards for growth in children. WHO percentiles can be used to address the stunted growth when it is less than 3rd centile\n\nC. Wasting: when Body Mass Index (BMI) is less than the 3rd centile for age or BMI Z score is less than 2 SD of the WHO Child Standards for BMI 5-19 years.\n\nD. Overweight: when Body Mass Index (BMI) is more than 85th centile for age or BMI z score is more than 1 SD of the WHO Child Standards for BMI 5-19 years.\n\nE. Obesity: when BMI is more than 97th centile for age or BMI Z score is more than 2 SD of the WHO Child Standards for BMI 5-19 years.\n\n2. Written informed consent was obtained from children parents or guardians accepting to participate in the study. Medical and sociodemographic data were recorded in the patient's chart.\n\n3. Children were examined clinically on dental units using artificial light. Source of it was magnifying loop with led light. Flat mouth mirrors and sterilized standard no.4/6 double-ended exploration probes. Wet cotton swabs were used prior to the examination to remove excess plaque or saliva.\n\n4. The diagnostic criteria for MIH and HSPM and scoring for them were established based on the European Academy of Pediatric Dentistry criteria7,8\n\n5. Caries Indices used in scoring dental caries; DMF (Permanent Teeth) where D = decayed indicated for restoration, M = missed due to caries, F = filled without recurrent caries. def (Primary teeth in mixed dentition) where d = decayed indicated for restoration, e= non-restorable indicated for extraction, f = filled without recurrent caries.\n\n6. Socioeconomic status: using a valid assessment tool. Socioeconomic status was measured using a tool developed and validated by El-Gilany et al., 2012. It measures the socioeconomic level through seven domains, education and cultural, occupation, family, family possessions, economic, home sanitation and health care domains. Total score of the tool is ranging from 0 to 84, with higher scores indicating better socioeconomic status.14\n\nSample size calculation\n\nThe sample size was calculated based on the primary objective in the study, which was detecting the prevalence of HSPM, MIH and dental caries in malnourished children. There was a previous study that estimated the prevalence of HSPM to be 5.6% and MIH to be 74%,15 while another former one estimated the prevalence of dental caries to be 83.1% in malnourished children.16 By setting the α as 0.05, power as 0.80, margin of error of 0.10 and the minimum required sample sizes for HSPM, MIH and dental caries is 21, 27 and 54 respectively. The largest sample size (54) was selected for the study.\n\nEfforts to address and avoid potential sources of bias\n\nWe excluded pediatrics with associated chronic illness as they might have additional external factors for malnutrition due to the chronic disease effect.\n\nData analysis\n\nAll statistical calculations were performed using SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 25, Categorical data was statistically described in terms of frequencies and percentages, while quantitative data were described in terms of mean and standard deviation, median, interquartile range and range as appropriate. Comparison of quantitative variables between the different groups of malnutrition was done using Kruskal-Wallis test as the variables were not normally distributed. Post hoc pairwise comparisons with Bonferroni adjustment of p value were performed between the groups. For comparing categorical data Chi square (χ2) test was performed, but obtaining p value was not applicable as 7 cells (> 25%) have expected count less than 5. For performing the correlation between two numerical variables, Spearman rho correlation was performed as the variables were not normally distributed.\n\nEthical considerations\n\nThe study was reviewed and approved by the scientific research committee and ethics of Cairo University, Faculty of Dentistry (ethical clearance number, 13321) and the study was carried out in accordance with Cairo University's laws human research. Throughout the study, the privacy and confidentiality of the data were preserved, the results were presented anonymously without disclosure of patients’ personal identifying information. Written informed consent for participation in the study and publishing was taken.\n\n\nResults\n\nThis study is a cross-sectional one which enrolled pediatric patients who were following in the outpatient dentist clinic across the duration of the study and observed to be malnourished for addressing the prevalence of HSPM, MIH and dental caries.\n\nTotal numbers of the study participants enrolled were 54 patients. The mean age was 7.10 ± 1.34. 50 % (n = 27) were males and 50% (n = 27) were females with male to female ratio 1:1 (Table 1).\n\n* SD: Standard Deviation.\n\nPrevalence of dental abnormalities (HSPM, MIH and dental caries) in the malnourished enrolled children were assessed (Table 2). It was 92.6% (n = 50) of the study group.\n\n* The remaining cases (total number 54) were not applicable.\n\nFor HSPM, its prevalence in malnourished children was 47.2% (n = 25) of the study participants. Regarding MIH, its prevalence was 45.2% (n = 19) of the malnourished enrolled study group. Lastly, the prevalence for dental caries was 64.3 % (n = 72) in the permanent teeth and 83.0 % (n = 44) in the primary teeth.\n\nThere was a co-occurrence between hypo-mineralized second primary molar and molar incisor hypo mineralization in the study group. It was 39% (n = 16) of the study group (Table 3).\n\nThe associations between the co-occurrence of HSPM and MIH and the HSPM, MIH and dental caries were examined. There were significant differences in the median HSPM for primary teeth, median MIH for permanent teeth and median CI (caries index) for primary teeth between patients who had co-occurrence and patients who didn’t. Median HSPM for primary teeth was 16.6% in patients who had co-occurrence of HSPM and MIH compared to 0% in patients who didn’t have this co-occurrence. Median MIH for permanent teeth was 20% in patients who had co-occurrence of HSPM and MIH compared to 0% in patients who didn’t. Also, CI for primary teeth was 7.4 in patients who had co-occurrence of HSPM and MIH compared to 4 in patients who didn’t (Table 4).\n\n* IQR: Interquartile Range.\n\n** P value is significant if < 0.05.\n\nMalnutrition in the enrolled children was assessed. The overweight group represented 24.1 % (n = 13). The obese group represented 16.7 % (n = 9). The underweight group represented 11.1 % (n = 6). The wasted group represented 18.5 (10 %). The stunted group represented 22 % (n = 40.7%) (Table 5).\n\n* Some cases showed more than one malnutrition disorder.\n\nTable 6 shows the associations between the types of malnutrition and the dental examination outcomes. Both HSPM and the MIH were significantly different among the types of malnutrition. For the HSPM, by performing post hoc pairwise comparisons with Bonferroni adjustment to the P value, it was found that HSPM was significantly different between the stunted group (median HSPM of 14.2%) and the overweight or obese group (median HSPM of 0.0%) (P value 0.01). Regarding the MIH level, by performing post hoc pairwise comparisons with Bonferroni adjustment to the P value, there were significant differences between the stunted group (median MIH of 19.4%) and overweight or obese group (median MIH of 0.0%) (P value 0.001), as well as between the stunted group (median MIH of 19.4%) and wasted groups (median MIH of 0.0%) (P value 0.025).\n\n* IQR: Interquartile Range.\n\n P value is significant if < 0.05.\n\n* The number and percentages of cases that showed the prevalence of co-occurrence of HSPM and MIH within each category of malnutrition.\n\n**** Chi square test is not applicable as 7 cells (> 25%) have expected count less than 5.\n\nCorrelations between dental abnormalities in the study group were assessed. MIH for permanent teeth showed a significant weak to moderate direct correlation with CI for primary teeth. HSPM showed a weak direct correlation with CI for primary teeth with borderline significance (Table 7).\n\n* P value is significant if < 0.05.\n\nThe Socioeconomic level of the study participants was investigated (Table 8).\n\n* IQR: Interquartile Range.\n\nThe most common socioeconomic level was the low one.\n\nThe associations between types of malnutrition of the enrolled children and their socioeconomic levels were investigated (Table 9). The highest score of the socioeconomic level was among the obese patients.\n\n* IQR: Interquartile Range.\n\n** P value is significant if < 0.05.\n\nThe association between the socioeconomic levels of the study group and the different dental abnormalities was investigated. HSPM was significantly different among the socioeconomic levels. By performing post hoc pairwise comparisons with Bonferroni adjustment to the P value, the HSPM showed a statistically significant difference between the very low group (median HSPM of 12.9%) and the high group (median HSPM of 0.0%) with P value of 0.017 (Table 10).\n\n* P value is significant if < 0.05.\n\n** IQR: Interquartile Range.\n\n\nDiscussion\n\nThis study is a cross sectional one which enrolled malnourished pediatric patients aged 5-10 years old who were following at dentist clinic at Cairo University for assessing the prevalence of HSPM, MIH and dental caries.\n\nRegarding the association between obesity or overweight group and the caries indices, our results agree with a previous one in which sensitivity analyses revealed that the obese children had more caries in their primary teeth than the normal-weight children. Considerably, more caries was noticed among the overweight and obese group in both the primary and the permanent teeth.17\n\nRegarding the association between MIH and the stunted growth, our results disagree with a previous study which showed that stunted schoolchildren had no significant correlation or association with MIH.18 This disagreement may be due to the high prevalence of short stature in the Egyptian children.19,20\n\nRegarding the association between socioeconomic level and obesity in the study participants, our results don’t agree with a previous study and found the higher prevalence of obesity in children with low socioeconomic levels. This different result may be due to different cultures.21\n\nRegarding the co-occurrence between HSPM and MIH, the present study goes in line with a former one that yielded similar results. That study concluded that HSPM can be considered a predictor of MIH.22\n\nRegarding the association between HSPM and caries index, HSPM showed a weak direct correlation with CI for primary teeth with borderline significance. This result is very close to a previous one in which an increased incidence of dental caries was found in children with HSPM.23\n\nRegarding the association between MIH and caries index, the present study shows a significant correlation. This result agrees with a previous study which yielded that caries values are much higher in children with MIH. However, it disagrees with it in that the total prevalence of MIH in that study was low unlike our study which showed a high prevalence of MIH as the study participants were malnourished. This reveals that malnutrition is a considerable risk for MIH.24\n\nRegarding the association between socioeconomic levels and the dental abnormalities, the present study showed higher percentages of MIH in lower socioeconomic levels. These results agree with a previous study which yielded lower socioeconomic levels in MIH.25 The present study slightly disagrees with a previous one in which caries index was higher in study groups with low socioeconomic levels and in which the socioeconomic level contributed no significant risk for HSPM.26 The latter disagreement may be due that in this study, the malnutrition was an added risk factor beside the low socioeconomic level.\n\nNot including controls of well-nourished children is considered as a limitation of the study. Not including controls of well nourished children in the study may make it impossible to detect whether the dental abnormalities are more prevalent in the malnourished children or not. Also, the non-probability sampling technique (consecutive sampling method) may compromise the generalizability (the external validity of the study) because it makes the study more liable for selection bias.\n\n\nConclusion\n\nMalnutrition could be a risk factor for dental abnormalities. Besides, children with low socioeconomic levels have a greater incidence for MIH. In addition, different dental abnormalities could co-exist together. Screening for HSPM, MIH and CI in malnourished children would be a welcome development.\n\n\nData availability\n\nFigshare. Childhood malnutrition and Hypomineralized molar defects, a cross sectional study. https://doi.org/10.6084/m9.figshare.16778557.v2.27\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\nBlack RE, Victora CG, Walker SP, et al.: Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet. 2013; 382: 427–451. Publisher Full Text\n\nGBD 2015 Child Mortality Collaborators: Global, regional, national, and selected subnational levels of stillbirths, neonatal, infant, and under-5 mortality, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016; 388: 1725–1774. Publisher Full Text\n\nAtef Abdelsattar Ibrahim H, Abdel-Raouf R, Zeid AS, et al.: Development of a simple and valid nutrition screening tool for pediatric hospitalized patients with acute illness [version 1; peer review: 2 approved, 1 approved with reservations, 1 not approved]. F1000Res. 2021; 10: 173. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOwlia F, Akhavan-Karbassi M-H, Rahimi R: Could Molar-Incisor Hypomineralization (MIH) Existence be Predictor of Short Stature?. Int. J. Prev. Med. 2020 Jul 9; 11: 101.\n\nHayden C, Bowler JO, Chambers S, et al.: Obesity and dental caries in children: a systematic review and meta-analysis. Community Dent. Oral Epidemiol. 2013; 41: 289–308. PubMed Abstract \| Publisher Full Text\n\nBagramian RA, Garcia-Godoy F, Volpe AR: The global increase in dental caries. A pending public health crisis. Am. J. Dent. 2009; 22: 3–8. PubMed Abstract\n\nWeerheijm K, Duggal M, Mejáre I, et al.: Judgement criteria for molar–incisor hypomineralisation (MIH) in epidemiologic studies: a summary of the European meeting on MIH held in Athens, 2003. Eur. J. Paediatr. Dent. 2003; 4: 110–113. PubMed Abstract\n\nElfrink ME, Ten Cate JM, Jaddoe VW, et al.: Deciduous molar hypomineralization and molar incisor hypomineralization. J. Dent. Res. 2012; 91: 551–555. PubMed Abstract \| Publisher Full Text\n\nAlaluusua S: Aetiology of molar–incisor hypomineralisation: asystematic review. Eur. Arch. Paediatr. Dent. 2010; 11: 53–58. PubMed Abstract \| Publisher Full Text\n\nFagrell TG, Ludvigsson J, Ullbro C, et al.: Aetiology of severe demarcated enamel opacities—an evaluation based on prospective medical and social data from 17,000 children. Swed. Dent. J. 2011; 35: 57–67. PubMed Abstract\n\nGhanim A, Manton D, Mariño R, et al.: Prevalence of demarcated hypomineralisation defects in second primary molars in Iraqi Children. Int. J. Paediatr. Dent. 2013a; 23: 48–55. PubMed Abstract \| Publisher Full Text\n\nPinto A, Kim S, Wadenya R, et al.: Is there an association between weight and dental caries among pediatric patients in an urban dental school?. J. Dent. Educ. 2007; 71(11): 1435–1440. PubMed Abstract \| Publisher Full Text\n\nOng DC, Bleakley JE: Compromised first permanent molars: an orthodontic perspective. Aust. Dent. J. 2010; 55: 2–14. PubMed Abstract \| Publisher Full Text\n\nEl-Gilany A, El-Wehady A, El-Wasify M: Updating and validation of the socioeconomic status scale for health research in Egypt. East Mediterr. Health J. 2012 Sep; 18(9): 962–968. PubMed Abstract \| Publisher Full Text\n\nChen D, Zhi Q, Zhou Y, et al.: Association between Dental Caries and BMI in Children: A Systematic Review and Meta-Analysis. Caries Res. 2018; 52(3): 230–245. Epub 2018 Jan 20. PubMed Abstract \| Publisher Full Text\n\nMittal N, Sharma BB: Hypomineralised second primary molars: prevalence, defect characteristics and possible association with Molar Incisor Hypomineralisation in Indian children. Eur. Arch. Paediatr. Dent. 2015; 16(6): 441–447. PubMed Abstract \| Publisher Full Text\n\nShakya A, Shenoy R, Rao A: Correlation between malnutrition and dental caries in children. Journal of Nepal Paediatric Society. 2013; 33(2): 99–102. Publisher Full Text\n\nOwlia F, Akhavan-Karbassi MH, Rahimi R: Could Molar-Incisor Hypomineralization (MIH) Existence be Predictor of Short Stature?. Int. J. Prev. Med. 2020 Jul 9; 11: 101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScreening and treatment of parasitic infestation, provision of iron/multivitamin supplementation and education on healthy nutrition should be part of school health programmes to prevent stunting in schoolchildren in Sohag.\n\nEl-Shafie AM, Kasemy ZA, Omar ZA, et al.: Prevalence of short stature and malnutrition among Egyptian primary school children and their coexistence with Anemia. Ital. J. Pediatr. 2020; 46: 91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang Z, Phung H, Hughes AM, et al.: Trends in overweight and obesity by socioeconomic status in Year 6 school children, Australian Capital Territory, 2006–2018. BMC Public Health. 2019; 19: 1512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNegre-Barber A, Montiel-Company JM, Boronat-Catalá M, et al.: Hypomineralized Second Primary Molars as Predictor of Molar Incisor Hypomineralization. Sci. Rep. 2016 Aug 25; 6: 31929. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHalal F, Raslan N: Prevalence of hypomineralised second primary molars (HSPM) in Syrian preschool children. Eur. Arch. Paediatr. Dent. 2020 Dec; 21(6): 711–717. Epub 2020 Apr 7. PubMed Abstract \| Publisher Full Text\n\nAhmad SH, Petrou MA, Alhumrani A, et al.: Prevalence of Molar-Incisor Hypomineralisation in an Emerging Community, and a Possible Correlation with Caries, Fluorosis and Socioeconomic Status. Oral Health Prev. Dent. 2019; 17(4): 323–327. PubMed Abstract \| Publisher Full Text\n\nWang L, Cheng L, Yuan B, et al.: Association between socio-economic status and dental caries in elderly people in Sichuan Province, China: a cross-sectional study. BMJ Open. 2017; 7: e016557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOyedele TA, Folayan MO, Oziegbe EO: Hypomineralised second primary molars: prevalence, pattern and associated co morbidities in 8- to 10-year-old children in Ile-Ife, Nigeria. BMC Oral Health. 2016; 16(1): 65. Published 2016 Jun 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtef Abdelsattar Ibrahim H: Childhood malnutrition and Hypomineralized molar defects, a cross sectional study. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "117504", "date": "05 Jan 2022", "name": "Osama El-Asheer", "expertise": [ "Reviewer Expertise Infant and pediatric clinical nutrition" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have reviewed the document from my Egyptian colleagues with great interest. This paper is well written and addresses an important topic which is the screening of dental abnormalities in malnourished children. The article is clear and of relevance also because there is a need for clinicians to develop an awareness about the possibility of dental abnormalities in the setting of malnutrition. It is today rarely described in international medical journals and there is no sufficient researches or data describing this relationship. Furthermore, the tool respects the WHO classification of malnutrition.\nAlthough the nutritional assessment here in the article depended only on the anthropometric evaluation, and others tools like the biochemical or dietary assessment are not present, there are several important conclusions from this article. For example, dental caries is a morbidity that could be found in obese children, and hypo-mineralization could be a morbidity in children with short stature. However, a correlation with obesity comorbidities could have been done to disclose more significance about the relationship between malnutrition and dental abnormalities. Very important: malnutrition could even represent a risk for the co-occurrence between MIH and HSPM. Correctly the authors excluded the chronic diseases to exclude bias as the chronic course could cause organic failure to thrive.\nTo improve paper readability, I suggest the following. Firstly, the discussion - although clear and focused - is better to be enriched with more related references. Secondly, the tables are better to be less, or summarized.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7770", "date": "14 Feb 2022", "name": "Hoda Atef", "role": "Author Response", "response": "Thank you dear reviewer for your valuable comments Point by point response to the comments 1. Reviewer's comment: Firstly, the discussion - although clear and focused - is better to be enriched with more related references Author's response: Done, I have enriched the discussion section by more than 30 updated references in the revised version 2. Reviewer's comment: the tables are better to be less, or summarized. Author's response: Done in the result section of the revised version" } ] }, { "id": "117506", "date": "10 Jan 2022", "name": "Huda Basaleem", "expertise": [ "Reviewer Expertise Community medicine and public health with subspecialty in 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\nThe topic is well chosen and addressing an interesting issue.\nTitle\nBetter to add Egypt (a cross-sectional study, Egypt).\nAbstract\nBackground: add “of the study” to the objective of the study.\n\nMethods: missing details about: sample technique (consecutive), period of the study, variables, and statistical analysis.\n\nKeywords: better to write the full name.\nIntroduction\nThe verbs of the objectives need to be amended:\nThe primary objective of the study is to estimate the prevalence……..\n\nThe Secondary objectives involve assessing the association between HSPM, MIH, and dental caries with co- occurrence of HSBM and MIH, and determining……….\nMethods\nThis section was written in a bulleted manner. The numbers 1-6 are not needed. Re-write\n\nPlease cite reference(s) for malnutrition definition.\n\nData “were” not data “was”.\nResults\nThe 1st paragraph is not needed (it is a repetition of the methodology).\n\nThe writing needs to be improved in many place. Avoid having small fragmented paragraphs like the description of tables in page 7.\n\nNo need to mention the M:F ratio as the sample are equally divided by gender.\n\nThere are 10 tables which are many for such a paper. Some tables are not needed like table 1, 3, 5 and 8. They are enough to be written as a text.\n\nThe description of the tables is misplaced. There is a possibility to put the writing about the table immediately before the table. The description of Table 10 is currently below the table; it should be before it.\n\nBetter to write the sample size in each title of the tables (n=54).\nDiscussion\nThis section needs to be totally re-written. It is now written with similar paragraphs and lacks the necessary depth of a sound discussion.\n\nInstead of writing “a previous study”, it is better to indicate which study: for example by the author, country or the year, or any other identification 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?\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": "7771", "date": "14 Feb 2022", "name": "Hoda Atef", "role": "Author Response", "response": "Thank you dear reviewer for your valuable comments Point by point response to the comments 1. Reviewer's comment : In the title section , Better to add Egypt (a cross-sectional study, Egypt). Author's response: Done in the revised version 2. Reviewer's comment: In the abstract section, Background: add “of the study” to the objective of the study. Methods: missing details about: sample technique (consecutive), period of the study, variables, and statistical analysis. Keywords: better to write the full name. Author's response: Done 3. Reviewer's comment, In the introduction part , The verbs of the objectives need to be amended: The primary objective of the study is to estimate the prevalence…….. The Secondary objectives involve assessing the association between HSPM, MIH, and dental caries with co- occurrence of HSBM and MIH, and determining………. Author's response: Done 4. Reviewer's comment In the methods parts, This section was written in a bulleted manner. The numbers 1-6 are not needed. Re-write Please cite reference(s) for malnutrition definition. Data “were” not data “was”. Author's response: Done 5. Reviewer's comment, In the result part, The 1st paragraph is not needed (it is a repetition of the methodology). The writing needs to be improved in many place. Avoid having small fragmented paragraphs like the description of tables in page 7. No need to mention the M:F ratio as the sample are equally divided by gender. There are 10 tables which are many for such a paper. Some tables are not needed like table 1, 3, 5 and 8. They are enough to be written as a text. The description of the tables is misplaced. There is a possibility to put the writing about the table immediately before the table. The description of Table 10 is currently below the table; it should be before it. Better to write the sample size in each title of the tables (n=54). Author's response: Done 6. Reviewer's comment The conclusion should have limitation and probably Recommendation. Author's response: Done 7. Reviewer's comment, In the discussion section, This section needs to be totally re-written. It is now written with similar paragraphs and lacks the necessary depth of a sound discussion. Instead of writing “a previous study”, it is better to indicate which study: for example by the author, country or the year, or any other identification information Author's response: Done, I have rewritten the discussion section and updated it" } ] }, { "id": "117503", "date": "17 Jan 2022", "name": "Hossam Abdelatty Eid Abdelmagyd", "expertise": [ "Reviewer Expertise Oral diagnosis", "oral medicine", "Periodontology", "Immunology", "medical education" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract\nThe comment written in the results part has to be rephrased to be statically significant mentioning p-value\n\nKeywords: no abbreviation in keywords is recommended\nIntroduction\nToo short introduction\n\nDefinition of malnutrition and its classifications have to be mentioned\n\nClassification of age groups based on WHO has to be added\n\nMethods\nStudy groups did not include a control group\n\nSample size not mentioned\n\nEthical approval of the institute not included\n\nExclusion criteria should include children with systemic diseases related to the digestive system\nResults\nThe statistical test used for data analysis is not mentioned\nDiscussion\nThe authors didn’t use numbers or percentages when comparing their study results with other studies which are very important for the quality of the discussion\n\nStudy limitations mentioned by authors themselves need to be addressed for validation of the results and 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? 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": "7772", "date": "14 Feb 2022", "name": "Hoda Atef", "role": "Author Response", "response": "Thank you dear reviewer for your valuable comments Point by point response to the comments 1. Reviewer's comment: In the abstract section, The comment written in the results part has to be rephrased to be statically significant mentioning p-value Keywords: no abbreviation in keywords is recommended Author's response: Done in the revised version 2. Reviewer's comment, In the introduction part, Too short introduction Definition of malnutrition and its classifications have to be mentioned Classification of age groups based on WHO has to be added Author's response: I have enriched the introduction with more information and details. Definitions of malnutrition and classification based on age by WHO have been addressed in the introduction with more details in the methodology as these classifications have been used in identifying malnutrition in the study group 3. Reviewer's comment In the methods parts, Study groups did not include a control group Sample size not mentioned Ethical approval of the institute not included Exclusion criteria should include children with systemic diseases related to the digestive system Author's response: One of the limitations of the study is that it didn’t include control groups. More studies are needed to address the difference between the malnourished and the well- nourished children regarding the dental abnormalities. There is a subtitle in the method section for sample size estimation and technique. There is a subtitle in the method section for the ethical approval; approved by the scientific research committee and ethics of Cairo University, Faculty of Dentistry (ethical clearance number, 13321) and the study was carried out in accordance with Cairo University's laws for human research. One of the exclusion criteria was children with chronic diseases which include diseases related to the digestive system 4. Reviewer's comment, In the result part, The statistical test used for data analysis is not mentioned Author's response: Written in the subtitle ( data analysis ) of the method section 5. Reviewer's comment, in the discussion The authors didn’t use numbers or percentages when comparing their study results with other studies which are very important for the quality of the discussion Study limitations mentioned by authors themselves need to be addressed for validation of the results and conclusion Author's response: I have enriched the discussion by more than 30 updated references with comparing the results. We agree with the reviewer regards the study limitation and it was described as a limitation. Any study has its own limitations, so further studies are needed to further address the possible extended outcomes of the research." } ] } ]	1	https://f1000research.com/articles/10-1307
https://f1000research.com/articles/10-867/v1	27 Aug 21	{ "type": "Research Article", "title": "Students’ perception of online learning amidst the Covid-19 pandemic: A study of junior, senior high school and college students in a remote area", "authors": [ "Senida Harefa", "Grace Lamudur Arta Sihombing" ], "abstract": "Background: The COVID-19 pandemic has brought about many changes in all sectors of life, especially in the field of education. These changes aim to make the learning process more effective in the pandemic environment. However, it can be challenging, as some students do not give positive responses to these changes, especially those in remote areas. This article aims to identify and report students' perceptions about the effectiveness of online learning during the COVID-19 pandemic in the remote North Tapanuli region of Indonesia. Methods: In this study, data were obtained using an online survey involving 30 students from three levels of education, namely junior high school, senior high school, and college. The data gathered from the survey were analyzed using quantitative descriptive methods. Results: Results show that online learning is considered less effective by students in remote areas; this happens because communication networks and infrastructure do not adequately support them to follow online learning. Conclusion: Teachers need to evaluate how to teach as well as re-design models and approaches to be applied in learning. This can be achieved by adjusting to the student’s current situation to generate interest and willingness to learn online.", "keywords": [ "students' perception", "online learning", "COVID-19 pandemic" ], "content": "Introduction\n\nThe COVID-19 pandemic has had a major impact on various aspects of peoples’ lives, namely in the economic, socio-cultural, and educational aspects. It is a global problem affecting educational institutions. Since the start of this pandemic, it has caused shock and disruption to students. The pandemic has forced schools to close and lessons that were carried out face-to-face have shifted to the online world. The use of the Internet and many other significant technologies to create materials for educational purposes, educational distribution, and program management constitute online learning (Fry, 2001). All educators are asked to make a transition, due to the closure of school buildings. There is no other choice but to apply online learning; even though many feel unprepared during this transitional period, students must adjust themselves while trying to build meaning amid various challenges related to the pandemic. Even though learning is carried out online, it is hoped that learning outcomes will remain maximal. There is some evidence that online learning can lead to higher student success (Kurucay & Inan, 2017). A great amount of evidence indicates that there is no substantial difference in the efficacy of well-designed online learning relative to well-designed face-to-face learning (Clark, 2007).\n\nHowever, the reality is not as expected since not all students respond positively to the implementation of online learning. Today, the majority of colleges and universities still face virtual learning difficulties (Talidong & Toquero, 2020). For example, not all educators and students can use e-learning applications, especially those in remote areas. They feel that they are not optimal in learning. During online learning, they deal with several obstacles such as more assignments that make them feel burdened. This happens since teachers or lecturers in charge assign them two or three tasks for every lesson. Additionally, network connection disturbance in rural areas affects their attendance of online learning. Online learning also influences the students’ motivation in doing assignments. Therefore, the objectives of online learning goals are not always achieved effectively. Students who succeed in learning are those who are active and always follow the learning. Parents of students also confirmed that their children were too lazy to learn online. This situation gives a bad picture of the learning attitudes of students.\n\nIn Anna Ya Ni’s research titled “A profile of MPA students’ perceptions of online learning: What MPA students value in online education and what they think would improve online learning experiences”, it is suggested that the use of the video chat software Zoom has the greatest potential to improve classes in order to meet student concerns. Zoom is one of the most frequently used applications in online learning to replace conventional face-to-face classes (Ni, Wart, Medina, Collins, Kimberly, & Pei, 2020) . The problems associated with online learning, especially in remote areas, motivated the authors to conduct this study. Therefore, this current study aims to identify the reasons why students in remote areas perceive that online learning during the COVID-19 pandemic is not effective.\n\n\nLiterature\n\nThe development of information and communication technology at this time provides many benefits for human life, so the mastery of such technology is no longer an option but has become a necessity. Through the existence of Internet networks, the use of technology in the educational environment has opened new avenues for educators; face-to-face learning has been transformed into e-learning or online learning (Bernard et al., 2009). In addition to other electronic media, such as CD-ROM, satellite, and television, some experts classify e-learning as ‘education delivered via the Internet’, while online education is described as ‘education delivered only via the Internet or web-based media’ (Lee, 2017). When used interchangeably, online education or e-learning is commonly defined as bridging the space between teachers and students through the use of web-based technology (Ryan & Young, 2015).\n\nThe presence of the Internet facilitates human work in many ways, especially in the field of education. The current learning process requires teachers and students to use technology. However, not all students can accept and adapt to these changes. The acceptance of changes in the learning process differs among students. This can be influenced by age, thinking ability, and students’ interest in technology. Students of all ages seem to react differently to the practice of online learning, with older students showing greater appreciation. There are still major variations in how learners view their online interactions during learning (Koohang, Paliszkiewicz, Nord, & Ramim, 2014). There are also concerns about the online learning environment’s efficacy (Hashem, 2011).\n\nStudents’ seriousness in taking online learning can be assessed by how they participate in ongoing learning. Participation in online learning requires three dimensions, namely cognitive participation, emotional participation, and behavioral participation (Fredricks, Blumenfeld, & Paris, 2004). These three dimensions are explained as follows: (1) Cognitive participation is the cognitive effort of a student to acquire skills in the online learning process. (2) Emotional participation is described as students’ positive emotions towards teachers, peers, and online learning. (3) Behavioral participation is participation that is manifested by activities that pay attention to learning when studying online (Jung & Jeongmin, 2018).\n\n\nMethods\n\nAn online-based questionnaire study was conducted in a remote area, North Tapanuli, Indonesia. The main objectives of this study were as follows:\n\n1. To assess students’ perception of the effectiveness of online learning during the COVID-19 pandemic using four indicators: 1) Teachers’ methods of online learning. 2) Students’ convenience in learning online. 3) Motivation to learn online. 4) The effectiveness of online learning.\n\n2. To find out the differences in average perception scores about online learning between three groups of students: 1) Junior high school students. 2) Senior high school students. 3) Students from college in a remote area.\n\n\nEthics\n\nThis research project was approved by the Research Ethics Committee. Ethical Approval Involving Human Respondent from tertiary education (Approval number: 1437.1/Ikn.01/TL.01/09/2020), from junior high school education (Approval number: 086/SMP-SM/IX/2020), and senior high school education, (Approval number: 422.1/063/SMA N 1TRT/2020). Written informed consent from all subjects involved was obtained for participation in the study and subsequent publication.\n\n\nData collection\n\nPrimary data was collected through an online survey (see Table 1). The survey included 20 items on a four-point Likert scale, from 1 (disagree), 2 (neutral), 3 (agree), 4 (strongly agree). The survey was conducted for over a week. Students were asked to participate in a web-based survey. Of the 75 students surveyed, only 30 students submitted their answers to the online survey, namely 10 students from junior high school education, 10 students from senior high school education and 10 undergraduate students from tertiary education. In this case, gender demography is an important factor to be analyzed.\n\n\nInstrument\n\nData in this study were collected through the use of questionnaires. Questionnaires consisted of four indicators; 1) Teachers’ methods of online learning; 2) Students’ convenience in online learning; 3) Motivation to learn online; 4) The effectiveness of online learning. Then the indicators were translated into 20 questionnaire items (Table 1).\n\n\nStatistical analysis\n\nData were collected, coded, checked for completeness and input into SPSS Version 25 IBM (SPSS Statistics, RRID:SCR_019096). R is an open-source alternative software that can also be used to do the same analysis. Descriptive statistics (frequency, percentage, mean and standard deviation) were used to describe variables. One-way analysis of variance (ANOVA) was used to determine differences in perceptual scores about learning online for junior high school, senior high school, and college students. In all experiments in this report, we applied an alpha level of.05.\n\n\nResults\n\nBased on the results of the calculation of the data obtained, the value of each questionnaire indicator was as follows: The teacher’s method in online learning (score = 89.8; average = 2,992; percentage = 74.83%), student comfort in online learning (score = 87.83; average = 2,928; percentage = 73.19%), learning motivation in online learning (score = 86.5; average = 2.883; percentage = 72.08%), effective online learning (score = 85.33; average = 2.846; percentage =71, 11%). After being calculated, the average percentage score = 72.96%. So, based on the hypothesis H1: p ≥ 85% (effective), H0: p ≤ 85% (less effective) indicates that students’ perceptions towards online learning in remote areas are less effective (Table 2).\n\nThe conditions that must be met to process data in a One-way ANOVA test are the data must be normally distributed, and the variance must be homogeneous. After our data were processed, the normality test met the first of these requirements, namely, a significance value of.103 > 0.05 (Shapiro-Wilk) thus the data was declared to be normally distributed (Table 3).\n\na Lilliefors significance correction.\n\nResults of the homogeneity of variance test obtained a significance value of.093 > 0.05. Thus, we can be confident that our data distribution was homogeneous (Table 4).\n\nThe output in the descriptive section shows the average value of students’ perceptions about online learning: student at junior high school (mean) = 58.10, student at senior high school (mean) = 55.30 and college student (mean) = 61.70. The highest score stating that online learning is less effective than face-to-face learning is that of college students, n = 30, 95% confidence interval for mean, total min = 48 and max = 73 (Table 5).\n\nThe ANOVA output in the descriptive section shows sum of squares total = 724,967; df = 29; mean square = 102,933 and 19.226; F = 5.354 and a significance value of 0.011 <0.05, meaning that the average value of student perceptions of the three levels of education about online learning is not significantly different. Students as a whole report the same perception that online learning is less effective in a remote area (Table 6).\n\nThen, the authors conducted a follow-up ANOVA test using Duncan’s test to determine the perceived significance value between junior high school with senior high school students and senior high school with college students. Duncan’s test results have two subsets, namely in the first subset, the significance value was 0.077 > 0.005 of senior and junior high school students’ perceptions, meaning that their perceptions about online learning are not significantly different. In the second subset, the significance value was 0.165 > 0.05 of senior high school and college students’ perceptions, meaning that their perceptions about online learning were also not significantly different. So, the results of Duncan’s test concluded that there was no significant difference between students’ perceptions of online learning, meaning that they had the same perception (Table 7).\n\nThe means of groups in homogeneous subsets are displayed.\n\nUses harmonic mean sample size = 10,000.\n\nIn this study, more male students answered that online learning was less effective than female students. The result of data calculation showed that the frequency of male students’ answers was 66.7%, while the frequency of female students’ answers was 33.3% (Table 8).\n\n\nDiscussion\n\nThe overall mean score obtained in this study p = 72,96% thus (H0:72, 96% ≤ 85%) indicates that students’ perception of online learning in remote areas is that it is less effective than face-to-face learning. The resulting score needs to be improved for the achievement of learning objectives. The indicators used to recruit perception data include the following:\n\n1. Teacher teaching methods in online learning\n\nThe use of effective learning methods or strategies can improve student academic achievement (Donker et al., 2013). Implementing learning, teachers must consider the use of teaching methods. The methods used should vary. Nowadays, technology offers a variety of learning methods that facilitate students to learn and do the assignments conveniently (Pasaribu et al., 2020). The application of various teaching methods can create creativity in learning and can eliminate boredom in students. There are five items regarding teaching methods, namely knowing, understanding, responding, describing and applying. The five items are inputs for the teachers so that they can design and review the online learning that has been implemented so far. The purpose of learning is to instill knowledge in students; whether good or not, students’ acceptance of the material presented depends on the method used by the teacher in learning. This also cannot be separated from the teacher’s own knowledge. The more knowledgeable a teacher is, the better he or she will be in conveying learning to their students.\n\n2. Comfort of students in online learning\n\nThe results of this study stated that students are less comfortable with online learning. The feeling of inconvenience represents dissatisfaction. For example, communicating with teachers is often hampered by unstable networks, and abilities in using the technology are still limited resulting in delays in joining lessons. Another regrettable thing is that most teachers do not understand the barriers that prevent the start of online learning or that affect it while it is ongoing. This may affect the effectiveness of online learning. In response to this, it is necessary to implement blended learning in the future, which combines online learning with traditional physical classroom teaching. It aims to enlarge the learning method in education areas. During the pandemic, the implementation of blended learning might occur in certain remote areas in Indonesia. The pandemic situation could be controlled because of the less density of population in remote areas and also by the strict application of health protocols: washing hands frequently, wearing masks all the time, and keeping a distance from one another (Garrison & Kanuka, 2004).\n\n3. Learning Motivation in online learning\n\nMotivation is the most important factor in learning. Motivation affects the achievement of student learning success and serves as an impetus to carry out learning activities. There are two types of learning motivation. The first is extrinsic motivation, which refers to all factors from outside that play a role in achieving learning goals such as facilities, teachers, and the process of implementing the learning. And intrinsic motivation is a factor from the students themselves such as interest, feelings of pleasure, and desire (Ryan & Deci, 2000). According to students in remote areas, online learning is less able to motivate students to learn. This is evidenced by students’ answers to the survey questions provided by the researchers. Students are not enthusiastic about online learning; they do not do assignments and do not submit assignments within the time that has been determined; they do not do study groups without the assistance of their teacher. This could be due to inadequate facilities, exhausted Internet packages or even students who cannot afford packages, and bad Internet network infrastructure. All these can cause a lack of motivation to learn in students. In summary, situations like this have a major impact on the way students learn and can lead to disappointing performance.\n\n4. Effective online learning\n\nIn this era, technology offers several advantages to assist human mobility practically. Besides, it also supports human communication and its efficiency, particularly the existence of cellular technology to facilitate rapid human connectivity (Song, Karimi, & Kim, 2015). During the COVID-19 pandemic, all schools in Indonesia used the Internet network to send messages to students (online learning). In other words, online learning tools that include technology support the independent learning process (Dunlap & Lowenthal, 2011). However, in using technology, it is also necessary to consider students’ perceptions. The results indicate that students in remote areas better recognized the effectiveness of face-to-face learning. As the aforementioned results related to the indicators suggest, students had several obstacles during online learning. The transformation of face-to-face into online learning affects the students’ learning process badly, for instance, the limitation of social interaction. In this case, the teacher is encouraged to think seriously about creative solutions to this problem to reach the teaching goals.\n\nFrom the frequency data, it is known that the response frequency was 66.7% by males and 33.3% by females, meaning that males responded more that online learning was not effective. Based on the results of data frequency, it is known that women’s motivation to learn online exceeds that of men. This is evident from the response of women to the tasks given by the teacher. In doing the tasks, women are much more disciplined than men. Also, women turn in assignments on time.\n\nThe COVID-19 pandemic presents an extraordinary situation worldwide, this situation affects the implementation of learning in schools. Face-to-face teaching and learning interactions turn to the online world. Given that not all students respond positively to online learning, each institution needs to prepare well for designing interesting learning media, and designing modules that are more flexible, making adjustments such that students adapt to changes in the teaching, learning and assessment, both face-to-face and online (Ansari et al., 2021).\n\n\nConclusions\n\nAccording to the previous explanations, students generally have more fun when learning is done face-to-face. With face-to-face learning, students can directly get answers to their curiosity about the material being studied. After conducting this research, the assumptions about the displeasure or reduced effectiveness of online learning in this area were proved correct and significant. Times have changed. With the COVID-19 pandemic, students and teachers are required to use technology in learning since learning must now be done remotely to prevent crowds from gathering, to break the chain of the pandemic. Willingly or unwillingly, online learning must be practiced. However, this is also a call for the government to improve Internet networks and infrastructure in remote areas in order to facilitate online education.\n\nThe results of this research indicate that online learning is less effective according to the perception of students in remote areas. So, educators are expected to redesign and implement procedures for online learning so that students can still learn as much as possible. From the teacher’s side, it is hoped that teachers will improve methods of teaching, by designing models and other approaches to provide variation in learning in order to raise students’ interest and willingness to learn online. To achieve higher levels of academic success, teachers must ensure there is a complete curricular plan that is tailored to goals, avoiding a large number of student burdens that are practically impossible to meet (Oliveira & Magalhães, 2020).\n\nThe results of this research provide additional insight to all those involved in the implementation of education. However, further research is needed to obtain a more complete explanation.\n\n\nData availability\n\nFigshare: Data survey about the effectiveness of online learning. https://doi.org/10.6084/m9.figshare.14191622.v1. (Harefa and Sihombing, 2021).\n\nThis project contains the following underlying data.\n\n• Research Data.xlsx (Questionnaire data in Microsoft Excel format)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAnsari KA, Farooqi F, Khan SQ, et al.: Perception on Online Teaching and Learning Among Health Sciences Students in Higher Education Institutions during the COVID-19 Lockdown – Ways to Improve Teaching and Learning in Saudi Colleges and Universities. F1000Res. 2021; 1–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernard RM, Abrami PC, Borokhovski E, et al.: A Meta-Analysis of Three Types of Interaction Treatments in Distance Education. Rev Educ Res. 2009; 1243–1289. Publisher Full Text\n\nCarr S: As Distance Education Comes of Age, the Challenge Is Keeping the Students. Chron High Educ. 2000; 39–41.\n\nClark RE: Reconsidering Research on Learning from Media. Rev Educ Res. 2007; 445–459. Publisher Full Text\n\nDonker AS, Boer Hd, Kostons D, et al.: Effectiveness of Learning Strategy Instruction on Academic Performance: a Meta-Analysis. Educ Res Rev. 2013: 1–26. Publisher Full Text\n\nDunlap JC, Lowenthal PR: Learning, Unlearning, and Relearning: Using Web 2.0 Technologies to Support the Development of Lifelong Learning Skills. In: E-Infrastructures and Technologies for Lifelong Learning: Next Generation Environments. USA: IGI Global; 2011; (pp. 292–315).\n\nFredricks JA, Blumenfeld PC, Paris AH: School Engagement: Potential of the Concept, State of the Evidence. Rev Educ Res. 2004; 59–109. Publisher Full Text\n\nFry K: E-learning markets and providers: some issues and prospects. Inf Discov Deliv. 2001; 233–239. Publisher Full Text\n\nGarrison DR, Kanuka H: Blended learning: Uncovering its transformative potential in higher education. Internet High Educ. 2004; 95–105. Publisher Full Text\n\nHashem ME: The role of new information technology in education and development: A case study of building trust in distance education. Int J Arts Sciences. 2011; 387–398.\n\nJung Y, Jeongmin L: Learning engagement and persistence in massive open online courses (MOOCS). Comput Educ. 2018; 122(7): 9–22. Publisher Full Text\n\nKoohang A, Paliszkiewicz J, Nord JH, et al.: Advancing a theoretical model for knowledge construction in e-learning. Online J Applied Knowledge Management. 2014; 12–25.\n\nKramer RM: Trust and distrust in organizations: Emerging Perspectives. Enduring Questions. Annu Rev Psychol. 1999: 569–598. Publisher Full Text\n\nKurucay M, Inan FA: Examining the effects of learner-learner interactions on satisfaction and learning in an online undergraduate course. Comput Educ. 2017; 115: 20–37. Publisher Full Text\n\nLee K: Rethinking the accessibility of online higher education: A historical review. Internet High Educ. 2017; 15–23. Publisher Full Text\n\nLuhmann N: Familiarity, Confidence, Trust: Problems and Alternatives. University of Oxford: Electronic edition, Department of Sociology, chapter; 2000; 6: 94–107.\n\nMaltby JR, Whittle J: Learning Programming Online: Student Perceptions and Performance. ASCILITE 2000 Conference. 2000.\n\nNi AY, Wart MV, Medina P, et al.: A profile of MPA students’ perceptions of online learning: What MPA students value in online education and what they think would improve online learning experiences. J Public Affairs Education. 2020; 1–23. Publisher Full Text\n\nOliveira RJ, Magalhães T: Teaching and learning based on peer review: a realistic approach in forensic sciences. F1000Res. 2020; 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPasaribu AG, Harefa S, Sihombing GL, et al.: Development Application of Web-Based Management of Scientific Work and Teaching Materials During the Covid-19 Pandemic. Solid State Technol. 2020; 1–17.\n\nRyan TG, Young CD: Online (Distance) Education: Evolving Standards. J Technologies Education. 2015; 15–30. Publisher Full Text\n\nRyan RM, Deci EL: Intrinsic and Extrinsic Motivations: Classic Definitions and New Directions. Contemporary Educational Psychol. 2000; 54–67. Publisher Full Text\n\nSims R: Rethinking (e)learning: a manifesto for connected generations. Distance Education. 2008; 153–164. Publisher Full Text\n\nSong D, Karimi A, Kim P: A Remotely Operated Science Experiment framework for under-resourced schools. Interactive Learning Environments. 2015; 1706–1724. Publisher Full Text\n\nTalidong KJ, Toquero CM: Philippine Teachers’ Practices to Deal with Anxiety amid COVID-19. J Loss Trauma. 2020: 1–8. Publisher Full Text" }	[ { "id": "94627", "date": "20 Sep 2021", "name": "Xuesong Gao", "expertise": [ "Reviewer Expertise 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\nIn the abstract, I am unsure if the changes brought by the pandemic were aimed to make the learning process more effective. I think that the background section needs to explain why the study needed to examine the students’ perceptions. I am not sure if 30 students were sufficient to examine the perceptions of students related to online learning at the three levels. The results are not particularly surprising. Since the number of participants in the study is quite small, I was wondering if the authors can make robust claims about the findings and these findings have significant implications for practice.\nIntroduction: I suggest that the authors summarize what was found in studies on students during the pandemic. A large number of studies have been published on the topic but it is true that not much was done in relation to students in remote areas. I do not think that the authors need to include the details of Anna Ya Ni’s research (I mean, the article title).\nLiterature review: Why do the authors talk about students’ seriousness in online learning? Not their commitment, engagement, or motivation? The literature review really needs to elaborate on why we need a study on students’ perceptions. It should also motivate the research questions to be addressed in the study.\nCan the authors explain their methodological decisions? I appreciate the challenge in collecting online responses. But I think that the authors need to acknowledge that the number of participants is a serious limitation. The authors may need to explain why the data were analyzed in the way reported in the manuscript. How do the collection and analysis of data correspond to the research questions? I think that we need an explicit alignment between research questions and data collection/analysis.\nI also suggest that the authors presented the results as answers to the research questions. This means that they can use the research questions to organize the presentation of findings. I am not sure if the authors need to follow APA to format the statistical reporting. I also feel that the authors need to explain what these statistical results mean. The manuscript has a lot of tables but it is better for the authors to engage with them and interpret the results for readers (as responses to the research questions).\nWhile the discussion has important themes, I feel that the authors need to highlight the contributions that the study has made to the field. In what sense do they add to what we have already known about the topic? Then they can talk about what implications we can draw from the findings.\nI feel that the authors need to discuss the study’s methodological limitations.\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": "93012", "date": "08 Dec 2021", "name": "Reza Rachmadtullah", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, the authors have done a great job. It is a very important topic under the current circumstances. I do not think that the authors need to include the title of Anna Ya Ni’s research in this article. There is general relevant information regarding e-learning during Covid-19 and is very current. In this study, it is better to add the latest references related to the research objectives in the discussion section. In the conclusion of this study, the authors should add a paragraph about the implications and usefulness of the results in this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7828", "date": "17 Feb 2022", "name": "Senida Harefa", "role": "Author Response", "response": "Dear Dr. Reza Thank you for your valuable suggestion to improve the study. We have modified the article as per your comments. Thank you again for your time as well. Best regards." } ] } ]	1	https://f1000research.com/articles/10-867
https://f1000research.com/articles/11-174/v1	11 Feb 22	{ "type": "Research Article", "title": "A randomised trial of the effects on recruitment and retention of including a wet-ink signature and photograph in the patient invitation letter for a clinical trial: results from a Study Within a Trial (SWAT 3 and SWAT 53)", "authors": [ "Rohan Anand", "Judy Bradley", "Brenda O'Neill", "Danny F. McAuley", "Mike Clarke", "Judy Bradley", "Brenda O'Neill", "Danny F. McAuley" ], "abstract": "Background: There is uncertainty about the effects of modifications to trial patient invitation letters on recruitment and retention. However, such documents are important. Methods: A 2x2 factorial design embedded a pair of randomised Studies Within a Trial (SWAT) within a large host trial (CLEAR), to evaluate the effects of including wet-ink signatures and photographs in the patient invitation letter. Patients were randomised to receive one of four invitation letters: with a wet-ink or generic signature, and with or without a photograph. The primary outcome was the proportion of invited patients who joined the CLEAR trial. The secondary outcome was the proportion of patients retained in the trial. Results: 368 letters were given to potential participants in the CLEAR trial and 121 (33%) joined. Proportions for each randomised group were generic signature and no photograph: 38% (33/88); generic signature and photograph: 32% (28/88); wet-ink personal signature and no photograph: 29% (26/91); wet-ink personal signature and photograph: 34% (34/101). There was no evidence of a significant difference in recruitment between those receiving the patient invitation letter containing a wet-ink versus generic signature (odds ratio (OR): 0.86, 95% confidence intervals (CI): 0.55 to 1.32, p=0.49) or photograph versus no photograph (OR: 0.99, 95% CI: 0.64 to 1.53, p=0.97). Retention was similar for the wet-ink and generic signature groups (OR: 1.20, 95% CI: 0.35 to 4.16, p=0.77) but significantly better when a photograph was used (OR: 5.40, 95% CI: 1.12 to 26.15, p=0.04, based on 2 withdrawals in the photograph group versus 9 in the no photograph group). Conclusions: These SWAT add to the evidence base for the effects of modifications to clinical trial documentation on recruitment and retention. We found including a photograph may improve retention. Although these analyses are underpowered, they will contribute to meta-analysis of similar comparisons. ISRCTN registration: 89040295 (06/07/2018)", "keywords": [ "SWAT", "Study Within a Trial", "Methodology", "Recruitment", "Retention", "Randomised trial", "Patient invitation letter" ], "content": "Introduction\n\nClinical trials depend on the willingness of a sufficient number of healthcare professionals and patients to dedicate their time and commitment to participate. Good recruitment is essential to the adequate conduct of a trial (Fisher et al., 2012) but is often difficult to achieve. For example, data from a review of large phase 3 randomised trials in the United Kingdom (UK) found that the recruitment target was met in only 56% of the trials (Walters et al., 2017). If the required levels of patient recruitment are not met, this has implications for the trial’s statistical power, likelihood of publication, validity, cost and duration, and leaves important questions about patient care unanswered (Glasgow et al., 1996; Bower et al., 2014). Currently, there are few evidence-based solutions to improve recruitment to trials and uncertainty about the effects of modifications to trial documentation (Treweek et al., 2018a), even though documents such as patient invitation letters feature in almost all trials. The Study Within a Trial (SWAT) approach for testing the effects of interventions that might improve recruitment or retention in clinical trials seeks to fill this gap by embedding a methodology study in an ongoing host clinical trial (Treweek et al., 2018b). Some SWAT use an observational design (Elfghi et al., 2020) while others, such as this, use a randomised design to allocate participants to different interventions (Bensaaud et al., 2020).\n\nThe invitation letter could influence whether a patient joins a trial. Different methods of personalisation, such as hand-written signatures by a member of the clinical research team or the inclusion of a friendly doctor photograph might affect patient recruitment, based on the mere-exposure effect or familiarity principle (Zajonc, 2001).\n\nOur aim was to explore if the type of signature or inclusion of a photograph in the patient invitation letter would impact on recruitment and retention in a clinical trial of treatment for bronchiectasis (CLEAR; ISRCTN 89040295, registered 6th July 2018; Bradley et al., 2019).\n\n\nMethods\n\nIn accordance with the ethical approval for these SWAT (North East - Tyne & Wear South Research Ethics Committee, reference number 17/NE/0339), informed consent was not obtained from the participants but written informed consent to join the CLEAR trial was obtained from the patients or their legal representative before they were randomised into the CLEAR host trial. The CLEAR trial was registered on ISRCTN (89040295) on 6th July 2018. This paper is reported in line with the Consolidated Standards of Reporting Trials (CONSORT) guidelines (Anand, 2021c).\n\nWe used a 2×2 factorial design to embed a pair of randomised SWAT in a large trial of treatment for bronchiectasis to evaluate the effects of including wet-ink signatures and photographs in the patient invitation letter. The host trial (CLEAR) was designed with Patient and Public Involvement (Bradley et al., 2019). In short, CLEAR is a yearlong trial where patients could be randomised to take a twice daily nebulised intervention along with daily tablets, weekly home spirometry and to attend five trial related visits. All patients who were potentially eligible for the CLEAR trial were eligible for the SWAT. The SWAT were registered in the SWAT Repository (Queen’s University Belfast, 2020). The first is a variation of SWAT 3 (Maguire & Clarke, 2014) and the second is SWAT 53 (Anand & Green, 2017). They were tested together in a 2×2 factorial randomised design.\n\nSWAT 3 relates to the nature of the signature on the invitation letter, with interventions being\n\n1. Invitation letter personally signed using wet ink by the local principal investigator.\n\n2. Invitation letter generically signed and printed electronically as “The CLEAR Trial Team”.\n\nSWAT 53 relates to the inclusion of a generic doctor-patient photograph in the invitation letter with interventions being\n\n1. Invitation letter includes a generic doctor-patient photograph.\n\n2. Invitation letter does not include a doctor-patient photograph.\n\nThe 2×2 design generated four different invitation letters (Table 1), which are available in the Extended data (Anand, 2021b).\n\nThe primary outcome of the SWAT is proportion of recipients who joined the CLEAR trial. The secondary outcome is the proportion of recruited participants who were retained in the CLEAR trial. These outcomes are taken from the routine data collected for the CLEAR trial.\n\nThe sample size for the SWAT is based on the sample size for the CLEAR trial.\n\nRecruitment packs were prepared centrally based on recruitment estimates for each site, with each pack having a unique Pack Identifying Number on the envelope containing the patient invitation letter. These Pack Identifying Numbers were randomly generated for each site using mixed block sizes and one of the four invitation letters was placed in each pack in accordance with this randomisation. Packs were prepared into bundles so that each bundle contained a random sequence of patient invitation letters and the bundles were sent to the sites with instructions on how to distribute them, along with other site initiation materials. Each pack contained an invitation letter, patient information sheet and informed consent form. The recruitment strategy for the CLEAR trial involved directly approaching potential participants who were regularly attending their respiratory clinic and giving them a recruitment pack. Potential participants were also identified within relevant databases and the recruitment pack was sent to them by post to their home address. Before the recruitment pack was given or posted to a patient, the Pack Identifying Number was recorded against the relevant Patient Identification Number on the screening log for the CLEAR trial. In this way, the randomised intervention was concealed from the patient and others involved in their recruitment until they had entered the SWAT. Participants could not be blinded to the SWAT intervention they received, but they were not aware of the SWAT or the hypothesis being tested.\n\nThe primary analysis compares the proportion of potential participants who joined the CLEAR trial in the randomised groups for wet-ink versus generic signature and for photograph versus no photograph. The secondary analysis compares retention in the CLEAR trial for these two comparisons. Analyses were done using the statistical software R (R Project for Statistical Computing RRID:SCR_001905; Version 1.2) and the statistical calculator on MedCalc (MedCalc Software Ltd. Odds ratio calculator. Version 20.023) was used to calculate the odds ratio (OR), associated 95% confidence interval (CI) and p-value for each comparison. The threshold for statistical significance was set to p=0.05.\n\n\nResults\n\nAll 14 sites in the CLEAR trial implemented the SWAT. The results presented here are final, as recruitment to the CLEAR trial was paused in March 2020 because of the coronavirus disease 2019 (COVID-19) pandemic and upon reopening in 2021 did not continue this SWAT substudy, but follow-up continued.\n\nAll analyses are based on data obtained between the opening of the CLEAR trial in June 2018 through to May 2020.\n\nIn total, 368 packs were given to patients across all sites (Anand, 2021a). The breakdown by type of invitation letter is shown in Table 2. A total of 121 (33%) of the invited patients joined the CLEAR trial.\n\nThe separate pairings were compared using odds ratios. Overall, no significant differences were found across the comparisons as shown in Table 3.\n\nAn odds ratio greater than 1 indicates that recruitment is more likely to occur in the first group listed in the comparison. CI=confidence interval.\n\n16 patients who joined the CLEAR trial and whose invitation letter had been randomised for these SWAT subsequently withdrew from the trial. Of these, 11 patients withdrew their consent under their own decision, while 5 were withdrawn for reasons outside the patient's control such as adverse events or decisions by their responsible clinician. Therefore, the analysis of retention uses data for a total of 110 (91%) patients: the 105 patients who remained in the CLEAR trial and the 5 who were withdrawn for reasons beyond their control (Table 4).\n\nRetention was similar for the wet-ink and generic signature groups (OR: 1.20, 95% CI: 0.35 to 4.16, p=0.77) but significantly better when a photograph was used (OR: 5.40, 95% CI: 1.12 to 26.15, p=0.04) a 12% difference in retention based on 2 withdrawals in the photograph group versus 9 in the no photograph group (Table 5).\n\nAn odds ratio greater than 1 indicates that withdrawal is more likely to occur in the first group listed in the comparison. CI=confidence interval.\n\n\nDiscussion\n\nThe analyses reported here did not reveal a significant difference in recruitment between the various types of invitation letter. However, this is not surprising given the relatively small sample size available for this analysis. For example, if 33% of the invited patients in a control group agree to join the trial, a sample size of approximately 3800 invitees would be needed to detect an improvement of 5% (to 38%), with 90% power at the 5% significance level. For the secondary outcome, one significant association was found, with patients who had received a letter that contained a photograph being more likely to remain in the CLEAR trial, however, this finding is based on small numbers and does not take account of the possibility of multiplicity affecting the likelihood of statistically significant results. If a trial retained approximately 90% of enrolled patients, a sample size of approximately 1200 recruited participants (3600 invitees) would be needed to detect an improvement of 5% (to 95%), with 90% power at the 5% significance level.\n\nThere are several factors to consider in these findings. Firstly, each SWAT tested minor changes to a small part of the overall recruitment pack that were in total 14 pages. Other information, such as the actual trial medication interventions, may have had greater influence on the decision made. Secondly, the intervention is non-verbal, while a large part of recruitment to trials involves verbal communication between trial staff and potential participants, so it is possible verbal discussions carry more influence especially in patients with chronic disease. Thirdly, the social psychological aspects of the mere-exposure effect can also work in reverse. For instance, rather than a patient having a positive preference for the familiarity of the wet signature and doctor photograph, they may not be happy with past experience of their clinical environment or care.\n\nThe analysis is consistent with the limited previous research into invitation material design. For instance, SWAT 3, 4 and 5 (Maguire et al., 2015) and SWAT 23 (Parker et al., 2018). Other studies also found limited differences in recruitment (Cockayne et al., 2017; Knapp et al., 2020; Man et al., 2015; Sheridan et al., 2020). However, one study (Gilbert et al., 2017) which aimed to enrol patients to a stop smoking programme, found that letters personalised to include an individual’s risk factors significantly increased participation, suggesting evaluating a more personalised approach for recruiting patients may have more impact than generic changes. Another study found handwriting the patient's name on the invitation letter, rather than printing it, decreased recruitment possibly due to handwriting being perceived as less professional (McCaffery et al., 2019). We note an ongoing study exploring invitation letter design on recruitment (Grønbech, 2018).\n\nStrengths of this analysis are that it is the first to simultaneously implement these two interventions in a 2×2 factorial design, with the use of allocation concealment and randomisation of the invitation letters. Overall, there were no major issues in implementation and the SWAT was administered with minimal cost. However, there were some imbalances in the types of invitation letters distribution (Table 2), likely due to mixed block sizes and the fact that sites were not recruiting patients at the same rate. Additionally, this SWAT did not encounter any of the issues regarding approvals, costs and site uptake encountered in some other SWAT (Martin-Kerry et al., 2019). The key limitation for this study is that it is an analysis based on convenience sampling. The CLEAR trial was paused in March 2020 due to the COVID-19 pandemic including all recruitment with a reopening planned for late 2021. It was anticipated that the revised recruitment strategy, that was primarily over the telephone, would alter the effect of the letters. Additionally, given the general challenges of re-opening recruitment and the revised time frame for trial completion, the management group agreed it would be beneficial for sites to focus their resources towards recruitment and answering the clinical questions of CLEAR. Based on the results of this analysis, continuing the SWAT in CLEAR would not alone detect an effect, therefore we hope this SWAT gives confidence to other trialists to replicate it in other studies.\n\n\nConclusions\n\nThese SWAT add to the evidence base for the effects of modifications to clinical trial documentation on recruitment and retention. Although these analyses are underpowered, they will contribute to meta-analyses of similar comparisons for this important question for clinical trials, as identified in the Prioritising Recruitment in Randomised Trials study (PRioRiTy I) (Healy et al., 2018).\n\n\nData availability\n\nZenodo: underlying data. https://doi.org/10.5281/zenodo.5792978 (Anand, 2021a).\n\nZenodo: Invitation letters design. https://doi.org/10.5281/zenodo.5654301 (Anand, 2021b).\n\nZenodo: CONSORT checklist for ‘A randomised trial of the effects on recruitment and retention of including a wet-ink signature and photograph in the patient invitation letter for a clinical trial: results from a Study Within a Trial (SWAT 3 and SWAT 53)’. https://doi.org/10.5281/zenodo.5824882 (Anand, 2021c).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank the CLEAR trial team at the Northern Ireland Clinical Trials Unit (NICTU) for help in the logistics and coordination of these SWAT and all sites that participated.\n\n\nReferences\n\nAnand R: underlying data [Data set]. Zenodo. 2021a. Publisher Full Text\n\nAnand R: Invitation letters design. Zenodo. 2021b. Publisher Full Text\n\nAnand R: CONSORT CHECKLIST V1. Zenodo. 2021c. Publisher Full Text\n\nAnand R, Green N: SWAT 53: Including a photograph on the invitation letter for a prospective study.2017. Reference Source\n\nBower P, Brueton V, Gamble C, et al.: Interventions to improve recruitment and retention in clinical trials: a survey and workshop to assess current practice and future priorities. Trials 2014; 15: 399. PubMed Abstract \| Publisher Full Text\n\nBradley JM, Anand R, O’Neill B, et al.: A 2x2 factorial, randomised, open-label trial to determine the clinical and cost-effectiveness of hypertonic saline (HTS 6%) and carbocisteine for airway clearance versus usual care over 52 weeks in adults with bronchiectasis: a protocol for the CLEAR clinical trial. Trials 2019; 20: 747. Publisher Full Text\n\nCockayne S, Fairhurst C, Adamson J, et al.: An optimised patient information sheet did not significantly increase recruitment or retention in a falls prevention study: an embedded randomised recruitment trial. Trials 2017; 18: 144. PubMed Abstract \| Publisher Full Text\n\nFisher L, Hessler D, Naranjo D, et al.: AASAP: a program to increase recruitment and retention in clinical trials. Patient Educ. Couns. 2012; 86(3): 372–377. PubMed Abstract \| Publisher Full Text\n\nGlasgow RE, Eakin EG, Toobert DJ: How generalizable are the results of diabetes self-management research? The impact of participation and attrition. Diabetes Educ. 1996; 22(6): 573–585. PubMed Abstract \| Publisher Full Text\n\nHealy P, Galvin S, Williamson PR, et al.: Identifying trial recruitment uncertainties using a James Lind Alliance priority setting partnership – the PRioRiTy (Prioritising recruitment in randomised trials) study. Trials 2018; 19: 147. PubMed Abstract \| Publisher Full Text\n\nKnapp P, Gilbody S, Holt J, et al.: Optimised patient information materials and recruitment to a study of behavioural activation in older adults: an embedded study within a trial. F1000Res 2020; 9: 9. Publisher Full Text\n\nMaguire L, Clarke M: SWAT 3: Gender of the person signing an invitation letter for a prospective study.2014. Reference Source\n\nMaguire L, Burns F, Clarke M: The NICOLA recruitment trial (NICOLA-RT): can you improve recruitment by making zero cost amendments to the invitation letter? Trials 2015; 16: P106. Publisher Full Text\n\nMan MS, Rick J, Bower P: Improving recruitment to a study of telehealth management for long-term conditions in primary care: two embedded, randomised controlled trials of optimised patient information materials. Trials 2015; 16: 309. PubMed Abstract \| Publisher Full Text\n\nMartin-Kerry J, Parker A, Bower P, et al.: SWATted away: the challenging experience of setting up a programme of SWATs in paediatric trials. Trials 2019; 20: 141. PubMed Abstract \| Publisher Full Text\n\nParker A, Knapp P, Treweek S, et al.: The effect of optimised patient information materials on recruitment in a lung cancer screening trial: an embedded randomised recruitment trial. Trials 2018; 19: 503. PubMed Abstract \| Publisher Full Text\n\nPROMETHEUS: PROMoting THE USE of SWATs. http\n\nQueen’s University Belfast: Studies Within a Trial (SWAT).2020. Reference Source\n\nSheridan R, Knapp P, Bower P, et al.: Patient recruitment to a diabetic retinopathy screening trial through optimised patient information materials: an embedded study within a trial (SWAT). F1000Res 2020 Jul 28; 9(779): 779. Publisher Full Text\n\nTreweek S, Pitkethly M, Cook J, et al.: Strategies to improve recruitment to randomised trials. Cochrane Database Syst. Rev. 2018a; 2018(2): MR000013. Publisher Full Text\n\nTreweek S, Bevan S, Bower P, et al.: Trial forge guidance 1: what is a study within a trial (SWAT)? Trials 2018b; 19: 139. PubMed Abstract \| Publisher Full Text\n\nWalters SJ, dos Anjos Henriques-Cadby IB , Bortolami O, et al.: Recruitment and retention of participants in randomised controlled trials: a review of trials funded and published by the United Kingdom Health Technology Assessment Programme. BMJ Open 2017; 7(3): e015276. PubMed Abstract \| Publisher Full Text\n\nZajonc RB: Mere exposure: A gateway to the subliminal. Curr. Dir. Psychol. Sci. 2001 Dec; 10(6): 224–228. Publisher Full Text" }	[ { "id": "137142", "date": "01 Aug 2022", "name": "Rustam Al Shahi Salman", "expertise": [ "Reviewer Expertise Stroke and clinical trial design/methods." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAnand et al. provide a very clear report of a well-designed (but imprecise) study within a trial (SWAT) of the effects on recruitment and retention of including a wet-ink signature and photograph in the patient invitation letter for a clinical trial. With a study sample size of 368, the SWAT did not find a significant difference in recruitment between those receiving the patient invitation letter containing a wet-ink versus generic signature, but they found that including a photograph in the invitation letter may improve retention.\n\nI have only a few suggestions:\nCould you put the article in the context of the PRioRiTy I and PRioRiTy II James Lind Alliance PSPs in the introduction?\n\nCould you provide 95%CI for the proportions described in the abstract to indicate the precision of the study?\n\nCould you provide the natural frequencies of the numerators and denominators and proportions in each group for the wet-ink versus generic signature and photograph versus no photograph comparisons in the abstract and Tables 3 and 5, for ease (to save the reader needing to calculate them themself)?\n\nElfghi et al., 2020, Bensaaud et al., 2020, Gilbert et al., 2017, McCaffery et al., 2019, and Grønbech, 2018 are not referenced.\n\nCould you give an indication of which ongoing RCTs are addressing SWAT 3 and SWAT 53, so that the reader understands to what extent and when these questions may be answered?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "151028", "date": "07 Oct 2022", "name": "Lydia O’Sullivan", "expertise": [ "Reviewer Expertise Clinical trial conduct", "clinical trial methodology", "evidence synthesis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting paper which describes a pair of SWATs evaluating the effect of using a wet-ink signature and a photograph included on invitation letters, on the recruitment and retention of participants to a respiratory trial. It’s great to see that 14 different trial sites carried out this SWAT and that both recruitment and retention were considered.\nThis is a concise and clearly written paper and the rationale for the SWAT is well described. I have made a few minor suggestions below:\nThe authors could consider clarifying in the Introduction, for those not familiar with the SWAT methodology, that SWATs are designed so as to not interfere with the outcome of the host trial.\n\nThe authors state in the Introduction that invitation letters feature in almost all trials. I worked for a number of years recruiting to and monitoring oncology clinical trials in an outpatient setting and invitation letters were rarely used – patients were approached with an information leaflet only. I’m not suggesting that invitation letters are not useful and clearly practices vary depending on the type of the trial, the requirements of the ethics committee, local operating procedures etc. But you might consider slightly re-wording this sentence.\n\nWho prepared the recruitment packs? Someone not involved in approaching potential participants?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-174
https://f1000research.com/articles/10-1024/v1	08 Oct 21	{ "type": "Research Article", "title": "Japanese sound-symbolic words in global contexts: from translation to hybridization", "authors": [ "Noriko Hiraishi" ], "abstract": "This paper explores the global reception and development of the artistic expression of onomatopoeia and mimetic words in modern and contemporary Japanese literary texts adopting the method of comparative literature. By analyzing sound-symbolic words and their translations in modern Japanese poetry and contemporary comics, the intercultural dialogues of these texts are examined and the emergence of hybrid onomatopoeia in global comic works is illuminated. The Japanese language is often noted for its richness of sound-symbolic words. In the literary world, modern poetry adopted and elaborated the use of these words from the late 19th century in its quest for a new style of poetry. In the early 20th century, poets developed the artistic expression of sound-symbolic words and succeeded in giving musicality to the “new-style poem”. However, the translation of Japanese sound-symbolic words has always been problematic. Experimental uses of these words in modern poems were often untranslatable, making the translations incomprehensible or dull. Nevertheless, graphic narratives and their worldwide distribution changed that situation. Japanese comics (manga) has particularly developed the artistic expression of sound-symbolic words. Usually placed outside speech balloons, these words are elaborately depicted and are important elements of the panel/page layout. Notably, the global popularity of the genre developed a new phase of intercultural dialogue. As not every word has an equivalent or is translatable in the target language, translators have left sound-symbolic words untouched in the translated versions, putting translation aside. Thus, the combination of Japanese and the target language seems to influence the visual comprehension of sound effects among the readers. Through the examinations of some cases, this paper brings to light the emergence of some hybrid onomatopoeia and reveals that the “Third Space” formed by the translation and hybridization of manga is a dynamic field that creates a new culture.", "keywords": [ "Sound-symbolic words", "Modern Japanese poetry", "Manga (Japanese comics)", "Translation", "Hybridization", "\"Third Space\"" ], "content": "Introduction\n\nOnomatopoeia and mimetic words have always colored our languages and literature. The Japanese language is often noted for its richness of these sound-symbolic words, and has around 4,500 of them (Ono, 2007). Sound-symbolic words are usually classified into three or five groups (Iwasaki, 2013: 69; Kindaichi, 1978: 5–8; Shibatani, 2006: 154) as follows:\n\n1. Phonomimes (onomatopoeia)\n\n➢ Animate phonomime (giseigo)\n\nvoice-mimicking words: words that mimic sounds made by living things\n\n➢ Inanimate phonomime (giongo)\n\nsound-mimicking words: words that mimic sounds made by inanimate objects\n\n2. Phenomimes (mimetic words)\n\n➢ Animate phenomime (gitaigo)\n\nmanner-mimicking words for living things\n\n➢ Inanimate phenomime (giyōgo)\n\ncondition-mimicking words for inanimate objects\n\n3. Psychomimes\n\n➢ Psychological/physiological-state-mimicking words (gijōgo)\n\nHowever, writers of the late twentieth century have not always appreciated this abundance of sound-symbolic words in the Japanese language. For instance, Yukio Mishima criticizes the use of onomatopoeia in fiction:\n\nOnomatopoeia brings daily conversation to life and gives it expressive power, but at the same time it typifies expression and makes it vulgar. (...) You will still find an onomatopoeia of laughter in popular literature, such as “All right, ha-ha…,” but everyone would be aware of the childishness of this technique.\n\n(Mishima, 1995: 140–141)\n\nAnother Japanese writer, Saiichi Maruya, takes a more neutral position, yet he also admits “the childishness” of sound-symbolic words1:\n\nJapanese language is abundant with these phonomimes and phenomimes. If you abuse them, you will give an impression of being childish, whereas it would be cold and hollow if you reject them strictly. (Maruya, 1977: 221)\n\nThis paper explores the artistic expression of onomatopoeia and mimetic words in Japanese literary texts. Although the stigma that renowned writers have placed on the use of sound-symbolic words has influenced Japanese fiction in the 20th century,2 modern Japanese poetry has cultivated such use of sound-symbolic words with a completely different attitude. Moreover, by considering sound-symbolic words in contemporary comics, this paper also reveals the contribution of sound-symbolic words to intercultural dialogue.\n\nRegarding Japanese comics in global contexts, the polysystem theory developed by Itamar Even-Zohar has contributed to deepening the debate on this issue (Rampant, 2010; Sell, 2011). As for the sound-symbolic words transcending language and culture, the discussion of the “Third Space” in recent translation studies also has great implications. Homi Bhabha argues that translation, as “the performative aspect of cultural communication” (Bhabha, 2004: 326), creates a “Third Space”, a boundary point where cultures collide and mix. Referring to Bhabha’s theories, especially those of “hybridity” and “in-between”, Michaela Wolf draws translation out of the bilingual problem into a different phase:\n\nIf we consider the Third Space as the potential and starting point for interventionist translation strategies, we realize that such strategies go far beyond the traditional concepts of “original” and “translation”, and the old dichotomy of “foreignizing” versus “domesticating” in all its implications. These strategies imply a shift toward the centre, where cultures encounter each other, and where meanings are effectively “remixed” (as shown in the example of liget). The place where cultures overlap and hybridity comes into being can already be considered as the locus of translation. This implies that culture is already itself translation. (Wolf, 2000: 141)\n\nFrom this perspective, translation “no longer bridges a gap between two different cultures but becomes a strategy of intervention through which newness comes into the world, where cultures are remixed” (Simon, 2000: 21). Adopting this point of view, this paper examines the intercultural dialogues of modern Japanese poetry and contemporary comics analyzing sound-symbolic words in these texts through translation, and argues for the emergence of some hybrid onomatopoeias in global comic works.\n\nThis study adopts the method of comparative literature, aiming to strike a balance between descriptive and interpretive case study approaches. The former approach implies historical and empirical knowledge, while the latter seeks to develop conceptual categories through close reading of the texts and to interpret the data. The paper, which is a series of case studies, does not allow us to draw any corpus-based conclusions, but it does allow us to identify the characteristics of each case and how it acquires meaning in different contexts. The data is extracted from published books, not from the first version published in magazines or other sources, except for the case of Indonesia, where several comic magazines have been published as a forum for the publication of original works.\n\n\n“Poem” and “Song”: The quest for musicality in modern poetry\n\nLet us briefly review the quest for a new style of poetry in modern Japan. Since Japan opened its ports to Western countries in the 1860s, the Japanese had been immensely impressed with and influenced by Western culture. Japan’s modernization was, as is often pointed out (e.g., Miyoshi, 1972), a cultural westernization. Art and literature played important roles in this process, sometimes prompting modifications and reconstructions of cultural memories, such can be seen in Japanese customs and the gap between the written and spoken languages. In the field of poetry, a movement to create a new form of Japanese poetry, abandoning the conventional Japanese formulas (5-7-5-7-7 and 5-7-5 syllable meters) and the Chinese-style, emerged under the influence of European literature. Shintaishishō (A Selection of Poems in the New Style), which sought to include some ideological and abstract content, incorporating stanzaic forms, rhymes and refrains, was published in August 1882. Translators who published translations of European poems also contributed to the development of shintaishi (new-style poem) as a new form of poetry.\n\nIt is worth noting that a fusion of poetry and western music was pursued in the process. Shūji Izawa introduced Shōka, a new word for song, to primary schools as an attempt to incorporate Western sounds into education. The three volumes of the Shōgaku shōka shū (Elementary School Shōka Collection) were published from 1881 to 1884, in which lyrics suitable to “cultivate virtue (Izawa)” (Tokyo University of the Arts and Centennial History Editorial Committee, 1987: 116) were put to the melody of hymns.\n\nFirst Be Fragrant\n\n1. Be fragrant, fragrant. Cherry tree of the garden.\n\n2. Stop, rest. Firefly on wild flowers.\n\n3. Wave, bend. Eulalia in the field.\n\n4. Cry, fly. Plover at the shallows. (lyrics by Chikai Inagaki)\n\nAs the first song of the collection’s first volume depicts the four seasons, its lyrics “incorporated plenty of poetic imagery of post-Man’yōshū (Collection of Ten Thousand Leaves) nature and human affairs in the Japanese islands” (Haga, 2002: 28). The lyrics follow the 2/4 time of the melody: ka/o/re/e, ni/o/e/e, so/no/u/no, sa/ku/ra/a.\n\nIn 1894, Tomoki Owada evaluated A Selection of Poems in the New Style and Elementary School Shōka Collection, stating that the former “pioneered the development of new-style poetry” (Santo, 2008: 150). He appreciated this work because it “tried to engender so-called Poemu (poem) in plain and simple words”, while the Elementary School Shōka Collection “was a model of so-called Songu (song) with lyrics often antiquated and old-fashioned”. Soon after this evaluation, shōka began to function as a device for “national education”. Bimyō Yamada was a poet who devoted his life to create shōka in the movement to unify the written and spoken styles of Japanese. His most popular shōka turned out to be a military song called Teki ha ikuman (Tens of Thousands of Enemies, 1891), with the melody composed by a professor at Tokyo Music Academy. Meanwhile, Omokage (Vestiges, 1889), an anthology of translated poems by Ogai Mori, among others, greatly influenced the literary world. The literati who absorbed Western culture through this anthology soon began to consider “music and poetry as an art” and tried to pursue the musicality of modern poetry. In a letter to his brother on November 29, 1894, Chogyū Takayama shared that in a concert he attended at Ueno Music Academy, where “piano, violin, shōka, military songs, sword dance, etc.” were played and performed, he was impressed with an art song called Autumn Breeze with lyrics by Bimyō Yamada:\n\nUntil today I thought that the shōka was a boring thing, but when I heard this, I realized that shōka could be a refined and elegant genre compared to Japanese music. (Takayama, 1933: 58)\n\nIt seems that his preference toward “refined and elegant” songs, which meant songs composed to good poems, influenced the literary world. We can see the continuance of this inclination in the 20th century, in a popular novel serialized in Yomiuri Newspaper in 1905: Fūyo Oguri’s Seishun (Youth). The novel starts with a recitation of a new-style poem, written by the main character, Kin’ya Seki. Praised highly by his friends, the poem was put to music and performed in a concert, which became a great success. Through the hero of this popular work of fiction depicted as a “new intellectual man”, we see that new-style poets at the time longed to have a song composed to one of their poems.\n\n\nModern poetry and onomatopoeia\n\nLet us now look at modern poets in this context. The movement to create a new form of Japanese poetry was also a quest for the prose poem, discarding or reorganizing the traditional 7-5 syllable meter. The reorganization of the 7-5 syllable meter was practiced by the early translators of European poems. Here is the first stanza of Paul Verlaine’s poem, “Chanson d’automne (Autumn’s Song, 1867)” and the Japanese version translated by Bin Ueda in 1905:\n\nLes sanglots longs Aki no hi no\n\nDes violons Violon no\n\nDe l’automne Tameiki no\n\nBlessent mon cœur Mi ni shimite\n\nD’une langueur Hitaburu ni\n\nMonotone. Uraganashi. (Verlaine,1962: 72, Ueda, 1978: 75)\n\nAlthough Ueda’s version is an adaptation rather than a translation, the rhythm of the poem is vivid in Japanese, using the repetition of a five syllabic meter. This version has been appreciated as a perfect example of “excellent translation” in the 20th century, with Donald Keene remarking: “But how much superior his Japanese version is to the English one!” (Keene, 1984: 227).\n\nAs a poet of keen senses, Hakushū Kitahara also applied his ingenuity to develop the sound and rhythm of his poems, adopting the meter of Imayō and Kouta. His first poem put to music was “Sora ni Makka na (In the Sky, Deep Red)” from his first collection Jashūmon (Heretical Faith) published in 1909.\n\nIn the sky, deep red are the clouds. Sora ni makka na kumo no iro.\n\nIn my glass, deep red is the whiskey, Hari ni makka na sake no iro.\n\nWhy do I feel so sad? Nande kono mi ga kanashikaro.\n\nIn the sky, deep red are the clouds. Sora ni makka na kumo no iro.\n\n(Translated by Margaret Benton Fukasawa, 1993: 36–37, Kitahara, 1984:29)\n\nIt is said that the members of “Pan Society” had chorused this poem to the melody of Rappa Bushi (Trumpet Tune) that was prevalent in the streets at the time (Nakamura, 1993: 96). Notably, the first music that Kitahara adopted for his poem was a popular song. The repetition of the beat 3, 4 and 5 in the rhythm of Imayō—an ancient verse form consisting of four lines each divided into two parts of seven and five syllables—convinces us that Kitahara was a poet interested in “singing poetry” from the beginning.\n\nCombining poetry and music, he made use of sound-symbolic words. Kitahara tried the artistic expression of sound-symbolic words in Heretical Faith and developed them in his later works, as a result of the achievement of musicality in poetry. In the poem “Sake to Tabako ni (With Wine and Cigarette)”, he uses three mimetic words effectively—uttori (enchanted), ukiuki (happily excited), and shikushiku (whimper)—making the best use of the repetition of the 7-5 syllable meter. These mimetic words not only made it easier to keep the 7-5 syllable meter, but also gave the poems a lively rhythm and a sense of visual dynamism through the use of hiragana script.3 Kitahara was a pioneer in using sound-symbolic words in his poems.\n\nHowever, the translation of sound-symbolic words has always been problematic, as the number and use of sound-symbolic words varies from language to language, and in many cases it is not possible to translate verbatim. Eugene Nida pointed out that in some languages “onomatopoeic expressions are considered equivalent to slang”, whereas they are “not only highly developed, but are regarded as essential and becoming in any type of discourse” (Nida, 2003: 169). Lafcadio Hearn translated some parody poems from Kyōka Hyaku Monogatari (A Parodic Poetry on Japanese Ghosts and Goblins), edited by Rōjin Tenmei (Old Tenmei) and published in 1853, as “Goblin Poetry” in The Romance of the Milky Way and Other Studies and Story (1905). He chose one poem with a peculiar onomatopoeia:\n\nTsuka-no-ma ni\n\nHari we tsutawaru,\n\nRokuro-Kubi\n\nKéta-kéta warau—\n\nKao no kowasa yo!\n\nSwiftly gliding along the roof-beam (and among the props of the roof), the Rokuro-Kubi laughs with the sound of “Kéta-kéta” —oh! the fearfulness of her face! (Hearn, 1905: 71)\n\nThe laughing sound of the long-neck goblin, Rokuro-Kubi, is not translated. Instead of translating the onomatopoeia, Hearn writes in the footnote: “‘Kéta’ means a cross-beam, but Kéta-kéta warau means to chuckle or laugh in a mocking way. Ghosts are said to laugh with the sound of Kéta-kéta”.\n\nThe translators of modern Japanese poetry have often followed Hearn’s way. Donald Keene translated Sakutarō Hagiwara’s “Neko (Cats, 1917)” as follows:\n\nTwo jet-black cats\n\nOn a melancholy night roof:\n\nFrom the tips of their taut tails\n\nA threadlike crescent moon hangs hazily.\n\n“Owaa, good evening.”\n\n“Owaa, good evening.”\n\n“Owaaa, the master of this house is sick.” (Keene, 1984: 268–269)\n\nHere, two cats meowing is the transliteration of the Japanese sound. Although Keene points out that “Hagiwara experimented with the musical values of the colloquial and of onomatopoeia” (Keene, 1984:267, he does not mention that this “Owaa” is not a common Japanese onomatopoeia for a cat meowing. Moreover, he misses the second last sentence of the poem, “Ogyaa, ogyaa, ogyaa,” another strange sound for a cat.4 It is questionable if English readers understand the quality of this poem. Although translators’ efforts have always been enormous, experimental uses of sound-symbolic words by modern Japanese poets have often been untranslatable, making the translations incomprehensible or dull. Here is another example:\n\nCircus (translated by Noriko Thunman)\n\nThere were several eras\n\nThere was a brown war\n\nThere were several eras\n\nIn winter the gales blew\n\nThere were several eras\n\nA brief flourishing—here, tonight\n\nA brief flourishing—here, tonight\n\nThe high rafters of the circus tent\n\nThere, one swing\n\nNot one you’d notice, a swing\n\nWith head upside down and hands hanging\n\nBelow the dirty cotton roof\n\nYuaan, Yuyoon, Yuyayuyon.\n\nAnd nearby, a white flame\n\nExhaling sharply like a cheap ribbon\n\nThe spectators are all sardines\n\nTheir throats whistle like the shells of oysters\n\nYuaan, Yuyoon, Yuyayuyon\n\nOutside it’s pitch black, the darkness of darkness\n\nThe long, long night deepens and deepens\n\nAlong with the nostalgia of the guy\n\nwith the parachute and\n\nYuaan, Yuyoon, Yuyayuyon (Thunman, 1983: 75–76)\n\nChūya Nakahara’s poem, “Circus”, was written in 1929 and published in his anthology Yagi no uta (Goat Songs, 1934). In addition to the poet’s particular attention to sentence layout (the translation follows the arrangement of the original), this poem is well-known for its experimental use of the phenomime “Yuaan Yuyoon Yuyayuyon”, a word coined by the poet which mimics the swinging trapeze. Tōru Kitagawa points out the importance of this mimetic word:\n\nThe image of the trapeze, swaying back and forth with the onomatopoeia “yuaan, yuyoon, yuyayuyon” under the dirty canvas of the tent, symbolizes the upset or crisis of Nakahara’s obstructed self-consciousness. This intense onomatopoeia moves in response to the swaying of an insecure feeling, rather than arousing the nostalgia. (Kitagawa, 1968:86)\n\nIt should be noted that the typography of the sound-symbolic words is also important in this case. Using the hiragana script ゆあーん　ゆよーん　ゆやゆよん, the poet depicts the stretching body of thee flying trapeze performer. It is not only the sound that matters here: the script type, the shape, and the visual effect are also very important. Unfortunately, the transliteration is not able to convey this information. The Roman transcription “yuaan, yuyoon, yuyayuyon” might be seen as a strange enumeration of sound. Consequently, another translator, Christian Nagle, adopted a more target-oriented sound-symbolic wording in a 2013 version: “see saw see and saw”.\n\nCircus (translated by Christian Nagle)\n\nfor a number of eras\n\nthere was a brown war\n\nfor a number of eras\n\na gale blew in winter\n\nfor a number of eras\n\nhere tonight a drinking party\n\nhere tonight a drinking party\n\nthere is a high beam in the circus tent\n\njust one trapeze\n\nan invisible trapeze\n\nunder soiled canvas of the big top\n\nhead down arms dangling\n\nsee saw see and saw\n\nclose to which white light\n\nexhales the words “cheap ribbon”\n\nthe audience are a bunch of sardines\n\nthroats gurgle oyster shells\n\nsee saw see and saw\n\noutside is dark dark on dark\n\nnight waning forever\n\nwith nostalgia for the damned parachutes\n\nsee saw see and saw (Nakahara, 2013)\n\n\nThe eloquence of sound-symbolic words in manga\n\nPerhaps the genre that best utilizes the visual appearance of sound-symbolic words in texts is comics. Onomatopoeia and mimetic words have always been a significant element of comics and graphic novels, because we read them as sound effects. As Ka-Boom!: A Dictionary of Comic Book Words, Symbols & Onomatopoeia (Taylor, 2007) shows, the comics genre has coined many new sound-symbolic words.\n\nJapanese comics, or manga, have particularly developed the artistic expression of sound-symbolic words. Usually placed outside speech balloons, these words are depicted elaborately though varying shapes, sizes, and texture. Manga artists have always tried to figure out ways of expressing onomatopoeia and mimetic words; a phenomime which represents “silence” was even invented in the 1950s (Tezuka, 1977: 108). Fusanosuke Natsume coined a word on’yu for the rich sound-symbolic words in manga, and states as follows:\n\nAs a result of diverse inventions and conversions, the onomatopoeia and its group in the manga actually even exceed the category of onomatopoeia—phonomimes/phenomimes/psychomimes— contributing to the “multi-layering” and “differentiation” of manga’s vocabulary. (Natsume, 1995:127)\n\nSound-symbolic words are considered one of the distinguishing features of manga. Figure 1 is from Naruto, Masashi Kishimoto’s blockbuster work. In this scene, the protagonist (Naruto) succeeds in his mission to ring the bell during a training session at the Ninja School, catching the instructor from behind. It is notable that the biggest space in this two-page spread panel is spared for the onomatopoeia ga!, the grabbing sound. Hinata (1986: 57) compares onomatopoeia in manga to sound effects in movies. Fukuma (1993: 190) points out that the “the size and font of hand-written onomatopoeia visually explain the volume of a sound, or the speed of an action”. The strong presence of sound-symbolic words in the panel expresses the energy of this scene.\n\n©2000 Masashi Kishimoto/Shūeisha. This figure has been reproduced with permission from Shueisha.\n\nAlthough hiragana and katakana have been the main writing devices used for the sound-symbolic words in manga, the modern English alphabet has also been used. While the use of Roman letters is not common in major Japanese manga compared to the cases in other countries, discussed by Valero Garcés (2008), we can find the ingenuity of some Japanese artists in words such as “BOMB!” (the sound of an explosion in English) in Akira Toriyama’s Dr. Slump (1981), or “FLOAT,” “BOOOM,” and “BOOO” in Kōhei Horikoshi’s Boku no Hero Academia (My Hero Academia, 2015). Furthermore, the sound-symbolic words in manga have created characters that were not originally possible. Yōji Yamaguchi points out the expressions of vowels with two dots (voicing mark) are popular in manga (Yamaguchi, 2014).\n\nNotably, the global popularity of the genre by digital diffusion5 developed a new phase of intercultural dialogues surrounding these words. It is easy to imagine that the translation of sound-symbolic words in manga can pose problems. However, various ways of translating have emerged, depending on target language and/or culture, or the policy of the publishers (Jüngst, 2008; Schodt, 2016: 7; Sell, 2011: 99–101). This paper hypothesizes that there are three ways thereof:\n\n1. Translation of all sound-symbolic words\n\n2. Preservation of the original expression and putting translation aside\n\n3. Leaving Japanese original sound-symbolic words untranslated, without any explanation\n\nAmong the collected examples analyzed for this paper, the first method seems to be the most common in English translations. French translations tend to adopt the second method,6 while the third method seems to apply with Chinese translations. However, further analysis and discussion is needed.7\n\nEarly examples from the last decade of the twentieth century show translators had found a way to translate phonomimes by changing their size, shape, font, and color. They often coined words as well. Heike E. Jüngst observes an intriguing case in German translation of Sadamoto and Gainax’s Neon Genesis Evangelion (1999) and states: “The use of onomatopoeia in translation can be very creative” (Jüngst, 2008). The translation of phenomimes was more difficult. Translators belabored to devise methods, as they sometimes could not find an equivalent word in the target language. James Rampant conducted an analysis of the English translation of Rumiko Takahashi’s Ranma 1/2 published in 1993 by Viz Media, where the pages were mirrored to change the reading direction from right-to-left into left-to-right, and the translators (Gerald Jones and Matt Thorn were credited for “adaptation”) sometimes changed or omitted the mimetic words. He pointed out that some phenomimes “have been translated with completely new dialogue, expansion, which is an example of the adaptation process that takes place in the production of the translation” (Rampant, 2010: 225).\n\nFor psychomimes, explanations are often added instead of translated. Previous studies have clarified that shōjo manga (a manga subgenre for girls) have made “discoveries of the inner self” (Otsuka, 1994) and have used multiple layers of language for elaborate psychological descriptions (Natsume, 1995; Otsuka, 1994; Yoshimoto, 2009). We can also find all the elaborated psychomimes to explain the characters’ feelings in this genre. In Karuho Shiina’s Kimi ni Todoke volume 1 (Hope it Reaches You, 2006), the emotional chemistry of the silent heroine is often told through phenomimes and psychomimes. As she is not good at expressing her emotions, Sawako, the heroine, often stares at someone with a blank expression or is moved by someone in secret. Ji, the phenomime for her stare, and jiin, the psychomime for being moved, are used repeatedly (Shiina, 2006). In the English version Kimi ni Todoke: From Me to You (2009), ji turns to STARE (Shiina, 2009: 14, 40, 129, 130, 131), and jiin is changed to SO HAPPY (ibid: 17) or to OVERWHELMED (ibid.: 17, 77, 120, 122, 124, 156, 165). In the French version Sawako (2010), ji is translated to REGARD FIXE (fixed gaze, Shiina, 2010: 14, 40, 129, 130, 131), while jiin turns to ÉMOTION (ibid.:17, 77) for the first two cases and ÉMUE (moved) for the rest (ibid.: 120, 122, 124, 156,165).\n\nIt is also worth noting here the cases of transliteration.8 In the French version of Gōshō Aoyama’s Meitantei Conan (Detective Conan; English version title is Case Closed) volume 61 (2010), many sound-symbolic words are transliterated: ban, a banging on the desk turns to BAM (Aoyama, 2010: 7); bin, the sound of stretching a string is VIM (ibid.: 17); a rupture tone pon is POM (ibid.: 27). The sound of a door opening is expressed in several different ways. Translator Misato Raillard translates the bigger sound gacha to CLAC and the smaller sound cha to TCHAC (ibid.: 123). The transliteration of the sound of a house burning in a fire, goooo (ibid.: 80) and dong (ibid.: 96), the sound of heavy objects being put down, is also interesting. Although these transliterations are not always adopted, we can find similar cases in other works.9 Moreover, French translations adopt the strategy to fit both the Japanese original text and the translation in the same panel. Japanese language learners may find this juxtaposition appealing.\n\n\nChallenges of global comics: The emergence of hybrid sound-symbolic words\n\nThis combination of Japanese writings (especially hiragana and katakana), sound, and the target language seems to influence the visual comprehension of sound effects among readers worldwide. The global influence of Japanese manga has grown considerably in the last decades of the 20th century. There are currently many manga-styled comic works in various countries,10 both in the form of magazines and books. In these works, we find some remarkable representations of sound-symbolic words, and some use Japanese sound-symbolic words. For example, Luca Molinaro and Giorgio Battisti juxtapose Japanese and Italian sound-symbolic words in their original Italian manga11 (Molinaro & Battisti, 2018). Loanwords and their derivations are also impressive. Sell (2011) pointed out the use of OHOHO, a feminine haughty laugh common in Japanese fiction, in Hollow Field (2007–2009), a work created by an Australian artist, Madeline Rosca (Sell, 2011: 99–100). Let us explore these cases in more detail. Odd Thomas is a thriller novel series written by Dean Koontz, first published in the United States in 2003. The series is about a 21-year-old short-order cook named Odd Thomas, who has the power to see the lingering dead. Following the success of the novels, three graphic novels and a movie have been released. The first graphic novel, In Odd We Trust (2008) was a collaborative work of Koontz and Queenie Chan, an Australian based manga-style comic book artist. In this work, we see some sound-symbolic words in Japanese: PU, to show Odd is blowing out (Koontz & Chan, 2008: 7, Figure 2), and SHU, the sound of cooking (ibid.: 12, Figure 3). Another noteworthy aspect of the use of these onomatopoeias is the way they are drawn. The addition of prolonged sound marks, which indicates a long vowel of two morae in length in Japanese, and the vertical writing are characteristic of the manga style. It is important to note that these expressions are acceptable to readers of the Odd series, who may not be particularly fans of Japanese manga.\n\nIllustration © 2008 Queenie Chan / Del Rey Books. These figures have been reproduced with permission from Queenie Chan.\n\nIllustration © 2008 Queenie Chan / Del Rey Books. These figures have been reproduced with permission from Queenie Chan.\n\nJenny’s Pink Diary was first published in 2005 as shōjo-manga styled BD in France, with the story set in Japan. The series won the 14th Anime & Manga Grand Prix of Animeland magazine, in the category “Best manga-styled BD” (Olivier, 2007). In volume 2 of this series, the sound of the classroom door opening is represented as CTHAC! (Jenny, 2006: 8, 15). This could be a derivative onomatopoeia of TCHAC as observed earlier. The volume shows another notable onomatopoeia: KIIYAAH (ibid.: 41, 72, 100) for a women’s scream, which reminds us of Kyaa, a common phonomime for women’s scream in Japanese. These examples anticipate the emergence of hybrid sound-symbolic words in the genre.\n\nThis hybridity of sound-symbolic words is also notable in Indonesia. Indonesia has a long history of comic works, and the influence of Japanese anime and manga on these works has grown in the late 20th century (Surajaya, 2010: 245). Manga-styled comic works in particular have developed in the country, with several magazines publishing original manga regularly.\n\nKyaa, the phonomime for a girl’s scream which was used in the aforementioned Pink Diary, is often seen in Indonesian comics in the form of KYAA, or KYAAA (Fauziyyah & Kurniawan, 2017: 63; Kartika, 2016: 50; Nisfihani, 2015: 42). Furthermore, we can also find a different usage of kyaa, as a phenomime expressing a woman being happy and talkative, in Indonesia (Viyanriri, 2017: 123; Zulvikar, 2016: 113).\n\nAnother interesting example is the onomatopoeic word HIKS. Annisa Nisfihani is an artist who became popular with her work Me vs. Big Slacker Baby, which depicts the delicate sensibilities of teenage girls in shōjo-manga style. Nisfihani colors her work with abundant phenomimes and psychomimes which are typical of Japanese girls’ comics. In chapter 6 of this work, published in 2016, we can see in the onomatopoeia HIKS HIKS to show a girl crying (Figure 4). This seems to be influenced by Japanese onomatopoeia hick hick, which usually depicts a child or female crying uncontrollably. What is noteworthy here is that HIKS seems popular in recent manga-styled Indonesian comics12 despite it being non-existent in Indonesian onomatopoeia. Thus, HIKS can be considered a hybrid onomatopoeia emerging from the comic genre in Indonesia.\n\n© 2016 Annisa Nisfihani/re:ON Comics. This figure has been reproduced with permission from re:ON Comics.\n\nTo understand the common usage of HIKS in this genre, we should consider the influence of the translation. Eriko Ono’s Kocchi Muite! Miiko (Look at Me! Miiko, 1995) has become one of the most popular manga for Indonesian girls through its animation series. The Indonesian translation of Ono’s manga was first published in 2002, and its long-lasting popularity led to the artist visiting Jakarta for a “meet and greet” event in 2013.13 The Indonesian version, translated by Widya Anggaraeni Winarya, has some interesting features concerning the sound-symbolic words. Winarya uses some fixed forms of onomatopoeia and mimetic words borrowed from or similar to Japanese, including HIKS. In the two scenes where a girl cries, the onomatopoeia HIKS is used in both cases (Ono, 2002: 103, 125). However, the original Japanese does not use hik in any of the scenes: rather, one scene uses gusu (Ono, 1995: 103), a mimetic word describing crying and sniffling, and the other uses jiwa (Ono, 1995: 125), a phenomime used to describe when tears start to flow. It seems the translator chose HIKS as a fixed onomatopoeia for a crying girl. Another case is the sound of the wind. In this volume, the cold wind in winter is expressed HYUUU (Ono, 2002: 151, 161, 162, 165). Although Hyu is actually a Japanese phenomime for the wind, the sounds byu (Ono, 1995: 151), byooo (Ono, ibid.: 161), byuu (Ono, ibid.: 162, 165) are used in the original. These choices made by translators may have been the foundation for the creation and the diffusion of hybrid sound-symbolic words in Indonesian original comics.\n\nThe hybridization of the appearance of sound-symbolic words is also evident. In Hiro Nurhadi’s Ankala, which was serialized in an Indonesian bi-monthly manga magazine, Shonen Fight, from 2015 to 2016,14 the artist uses a distinctive font for the sound-symbolic words (Nurhadi, 2015: 222–245). The font reflects katakana-inspired fonts, such as Tokyosoft created by Shrine of Isis in 1998, or Electroharmonix published by Ray Larabie in 2015. The technique of using stylistic fonts to design pages has also been adopted by French artist Tony Valente, in volume 8 of Radiant (2017).\n\n\nConclusion\n\nAlthough Yukio Mishima denounced the use of onomatopoeia as a “childish technique” in the 1950s, modern Japanese texts have persistently developed the utilization of sound-symbolic words. Modern poetry played a leading role in this as poets in the early 20th century attempted to create new-style poetry. The pursuit of musicality in modern poetry inspired poets’ artistic use of onomatopoeia and mimetic words. Mixing the scripts and sometimes inventing new sound-symbolic words, they succeeded in giving the new-styled poem a fresh rhythm and visual effect. However, the translation of Japanese sound-symbolic words has always posed a challenge as experimental uses of these words in modern poems were often difficult to translate.\n\nIt was graphic narratives and their worldwide distribution that broke through that situation. In this genre, we recognize sound-symbolic words not only as sound effects but also part of the picture, an element inseparable from the story. This recognition invites the reader to take an interest in Japanese sound-symbolic words, and the ingenuity and challenges of the translators are widely appreciated with diffusion through the readership. The parallel notation of Japanese and target languages in the translation of sound-symbolic words deserves special mention. This method has influenced the visual comprehension of phonomime, phenomime, and psychomime among readers worldwide, and created novel expressions; hybrid sound-symbolic words. The cases of emerging hybrid onomatopoeia and mimetic words analyzed and discussed in this paper clarified this process, pointing to the growing importance of sound-symbolic words in the genre, depicted in a variety of ways in comics around the world. They also reveal that the “Third Space” formed by translation and hybridization of manga is indeed a dynamic field that creates a new culture. It is worth examining these words in the genre as they continue to evolve, and we can expect more stimulating examples.\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": "Footnotes\n\n1 Valero Garcés (2008) introduces the argument of Michaela Schnetzer that “the use of onomatopoeia is associated with genres (e.g. comic books, cartoons, children’s literature) that are still considered by a large sector of the general public as not ‘serious’ enough to be the subject of academic research.”\n\n2 Notably, when Banana Yoshimoto made a striking debut in the 1980s, her style with abundant sound-symbolic words was undervalued by many critiques as “girls’-comic-like style.” (Hara, 2012:53)\n\n3 The Japanese writing system is a combination of the logographic kanji (Chinese characters) and the syllabic kana (hiragana and katakana). Katakana used to be the script for foreign words, loanwords, and onomatopoeia.\n\n4 “Ogyaa” is usually used as an onomatopoeia for a baby’s cry.\n\n5 See Robin E. Brenner (2007) Understanding Manga and Anime, Westport: Greenwood Publishing, Federico Zanettin (ed.) (2008) Comics in Translation, London: Routledge, Toni Johnson-Woods (ed.) (2010) Manga: An Anthology of Global and Cultural Perspectives, New York: Continuum. This situation has given rise to the important topic of “scanlation,” but for reasons of space, this paper will not deal with this subject.\n\n6 Celotti (2008) observes a case of French translation of an Italian comic, where the onomatopoeia “is left unchanged”.\n\n7 At this point, we have only analyzed a few cases and cannot determine if there are any differences due to language or culture. We are planning to proceed the research with more extensive data collection and analysis.\n\n8 Regardless of the subgenre (boys or girls, etc.), many translations in French have adopted this strategy.\n\n9 In Sawako, door-opening sound gacha turns to GATCHAK.\n\n10 There are terms for the genre, such as “Original Non-Japanese Manga (ONJ manga)”, (Sell, 2011: 94), and “pseudomanga” (Jüngst, 2008).\n\n11 The series is published in the category “manga.”\n\n12 We’ve found the use of “hiks” in Anisah Fauziyyah and Adhi Kurniawan, The Witness Mirrors (2017:46), Gladys E (Gladys Elisabeth), My Dear Janitor (2017: 75), Thoma & Herrad (Thoma Prayoga and Herrad Syafrian), The Story about My Father (2017: 159, 169), and Archie the Redcat, Mulih (2017: 23).\n\n13 Chao, October 2013, p.104. https://www.antaranews.com/tag/hai-miiko\n\n14 In the course of this research, we learned that Mr. Hiro Nurhadi passed away on May 11, 2016. 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Publisher Full Text\n\nSanto I: Shōka to kokugo: Meiji kindaika no sōchi. (Shōka and National Language: A Device for Modernization), Tokyo: Kōdansha. 2008. Reference Source\n\nSell C: Manga Translation and Interculture. In Mechademia: Second Arc. Minneapolis: University of Minnesota Press, 2011; 6: 93–108. Publisher Full Text\n\nShibatani M: The Languages of Japan. Cambridge: Cambridge University Press, 2006[1990]. Reference Source\n\nShiina K: Kimi ni todoke. Tokyo: Shūeisha. 2006; 1.\n\nShiina K: Kimi ni Todoke: From Me to You. translated by Tomo Kimura. San Francisco: Viz Media, 2009; 1. Reference Source\n\nShiina K: Sawako. translated by Pascale Simon. Bruxelle: Kana. 2010. 1.\n\nSchodt FL: Translating Manga. In World Literature Today,. March/April 2016, Norman: University of Oklahoma, 2016; 90(2): 7.\n\nSimon S: Introduction. In Sherry Simon and Paul St-Pierre (eds) Changing the Terms: Translating in the Postcolonial Era. Ottawa: University of Ottawa Press, 2000; 9–29. 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(The Centennial History of Tokyo University of the Arts), Tokyo ongaku gakkō hen (Tokyo College of Music,), Tokyo: Ongaku no tomo sha. 1987; 1.\n\nToriyama A: Dr. Slump. Tokyo: Shūeisha. (2012[1981]); 5. Reference Source\n\nUeda B: Teihon Ueda Bin zenshū. (Complete Works of Ueda Bin), Tokushima: Kyōiku Shuppan Center. 1978; 1.\n\nValente T: Radiant. Roubaix: Ankama. 2017; 8. Reference Source\n\nValero Garcés C: Onomatopoeia and Unarticulated Language in the Translation and Production of Comic Books. In Federico Zanettin (ed.) Comics in Translation. Oxon and New York: Routledge, ebook. (2014[2008]).\n\nVerlaine P: Oeuvres poétiques completes (Complete poetic works). Paris: Gallimard, 1962.\n\nViyanriri R: Things Happened. In: Permen: Kompilasi Komik Koloni Bergaransi. (Candy: Compilation of Guaranteed Comics). Jakarta: M&C, Gramedia, 2017; 121–150.\n\nWolf M: The Third Space in Postcolonial Representation. In in Sherry Simon and Paul St-Pierre (eds) Changing the Terms: Translating in the Postcolonial Era. Ottawa: University of Ottawa Press, 2000; 127–145. Reference Source\n\nYamaguchi Y: Tenten: Nihongo kyūkyoku no nazo ni semaru. (Double Dots: The Ultimate Mystery of the Japanese Language), Tokyo: Kadokawa shoten, ebook. 2014.\n\nYoshimoto T: Zen manga ron. (Studies on Manga) Tokyo: Shōgakukan. 2009.\n\nZulvikar F: Wanoja. chapter 7, In Kosmik Mook. (Cosmic Mook) Jakarta: M&C, Gramedia, 2016; 9: 105–122." }	[ { "id": "96576", "date": "10 Nov 2021", "name": "Takayuki Yokota-Murakami", "expertise": [ "Reviewer Expertise Comparative literature", "Japanese popular culture" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 gives an interesting account of the onomatopoeia in Japanese literature and comics and their significance in the instancing of “the Third Space” in the global world culture (in the case of comics). The author attempts to locate the problems with the contemporary theoretical framework, at the same time giving analyses of many intriguing cases in which onomatopoeia are negotiated and “translated” in the comic media. The paper deserves to be indexed, but for the shortcomings listed below, it needs extensive revision.\nThe shortcomings of the paper, to my view, are as follows:\nThe paper consists of three sections: a theoretical introduction to the problems of onomatopoeia in Japanese; their historical trajectory in the Japanese modern poetry (mostly from the Meiji period); and the analyses of “translational” use of onomatopoeia in the translation of Japanese manga in the global market. However, the connection among those sections is quite weak. The author mentions in the conclusion that translation of Japanese manga has demonstrated the creative and translational potential of onomatopoeia which was suggested by the poetic experiments in the modern Japanese literature, but the historical trajectory of such a development is not described, that is to say, the connection between them remains purely theoretical. But as the theoretical analyses of onomatopoeia remains under-developed and largely haphazard (for instance, the distinction among the various kinds of onomatopoeia in the linguistical study in the first section is almost neglected in the third section), readers might question the necessity of the first and the second sections. The concept of “musicality” presented in the second section is not fully fledged either (and the readers wonder why the concept has to be brought out).\n\nThe third section gives interesting and more or less convincing arguments on the various strategies of translating onomatopoeia in world comics. However, whether they constitute, as the author claims, “the Third Space” (which I might term “translational zone” following Emily Apter1) remains somewhat polemical. One might wonder if the cases given are those of adaptation or influence rather than the emergence of “the Third Space.”\n\nThe case analyses given in the third section, quite stimulating though they are, are mostly taken from the existing previous researches. The author should present more of their own research results.\n\nFinally, occasional grammatical and stylistic errors such as “proceed (promote?) the research” (endnote 7).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7788", "date": "11 Feb 2022", "name": "Noriko Hiraishi", "role": "Author Response", "response": "Thank you very much for your very useful comments. I will keep your comments in mind for future work and will further deepen my research. Most of the case studies in the third section are the results of the author's own research, not the results of previous studies. I would appreciate your understanding on this point." } ] }, { "id": "96578", "date": "17 Nov 2021", "name": "Rima Devi", "expertise": [ "Reviewer Expertise Literature", "Japanese literature", "and Japanese Language 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\nIn general, this article is interesting because it explains how the translator attempts to translate Japanese into the target language. The translation conveys messages from the source language to the target language: \"Communicative translation is social, concentrates on the message and the main force of the text, tends to under-translate, to be simple, clear and brief, and is always written in a natural and resourceful style\" (Newmark, 1988: 47-48). There will always be words from the source language that have no equivalent in the target language. Translators try to convey messages from the target language even by removing, adding, or creating new vocabulary in the target language. This translation process makes translators of literary works considered as creators of new works in the target language. In translating poetry and song lyrics, the creation of new words is considered normal because the authors of poetry and song lyrics have been accepted in society as a group that violates standard grammar rules. It can be seen in the poems of Shakespeare (English poet) and Khairil Anwar (Indonesian poet).\nWith the creation of new vocabulary by translators when translating the manga into Indonesian, for example, it cannot simply be said that there has been hybridization in translation. New vocabulary is created because there are no equivalent words for onomatopoeic and mimetic words in Indonesian. Meanwhile, in Japanese, there are many onomatopoeic and mimetic words.\nThe hybridization proposed by Bhabha (2004) occurred because of colonialization, and the colonized were marginalized and had no place to actualize themselves. Hybridity refers to the creation of new transcultural that exist within the confluence area resulting from colonization: \"One of the most widely employed and most disputed terms in postcolonial theory, hybridity commonly refers to the creation of new transcultural forms within the contact zone produced by colonization. As used in horticulture, the term refers to the cross-breeding of two species by grafting or cross-pollination to form a third, ‘hybrid’ species\" (Ashcroft et al., 2013: 135-136). Meanwhile, in the translation from Japanese to Indonesian, has colonialization caused this translation? This matter needs further discussion.\nIt is too early to conclude that there has been hybridity in manga translation. More accurate data are needed to prove whether hybridization has occurred, and there needs to be adequate evidence and analysis that the third space is a means to publish translated works such as manga.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7793", "date": "11 Feb 2022", "name": "Noriko Hiraishi", "role": "Author Response", "response": "Thank you for your valuable comments. I will send you the revised manuscript." } ] } ]	1	https://f1000research.com/articles/10-1024
https://f1000research.com/articles/11-172/v1	11 Feb 22	{ "type": "Research Article", "title": "Effect of microfluidic rectangular microelectrode geometry on bioparticles manipulation in dielectrophoretic application", "authors": [ "Zuriel Da En Shee", "Ervina Efzan Binti Mhd Noor", "Aminuddin Bin Ahmad Kayani", "Noor Ziela Binti Abd Rahman", "Zuriel Da En Shee", "Aminuddin Bin Ahmad Kayani", "Noor Ziela Binti Abd Rahman" ], "abstract": "Background: Microfluidic cell manipulation techniques have been continually developed and integrated into miniature chips as a so-called lab-on-a-chip (LOC) platform for high-throughput bioassays. Among the various mechanisms of bioparticles manipulation by electrically induced forces, dielectrophoresis (DEP) has been regarded as the most promising technique utilized in microfluidic systems. Into the micro- to nano-scale level of DEP configuration, the common challenges of undesirable side effects such as electrohydrodynamic effects, joule heating, and electrolysis that may occur in the microfluidic system has always been a hurdle which would severely limit the DEP performance. Methods: A numerical simulation study was performed on a versatile capability of a rectangular type of dielectrophoresis microelectrode with different parametric design configuration variables (channel height: 20-50 µm; electrode width 20-100 µm; electrode spacing 5-50 µm). Results: Numerical study results have shown that the ideal dimension range of design configuration for optimum DEP performance have been identified to be 40µm in channel height, 20-40 µm in electrode width and 5-15µm in electrode spacing, further increasing of the dimensions have shown a decrease in DEP performance consequently abridged the bioparticle manipulation. Conclusion: This investigation of the parametric design of the rectangular geometry microelectrode has provided necessary insight to the microelectrode design information and parametric considerations for optimum DEP device fabrication and enhancement.", "keywords": [ "Microfluidic", "electrophoresis", "dielectrocphoresis", "geometry", "microelectrode", "protein" ], "content": "Introduction\n\nDielectrophoresis (DEP) has been proven as a versatile manipulation technique commonly used in a microfluidic platform1. DEP technique is commonly used to manipulate dielectric particles in a non-uniform electric field by the induction of DEP force (FDEP)2. Dielectrophoresis is an electrokinetic motion phenomenal occurring when a polarizable particle is put in nonuniform electric fields, and the particle mobility is influenced by the ambient electric field, as well as the characteristics of the dielectric particles or solutions3. Its versatility has provided various benefits for clinical application, laboratory diagnostics and research application4.\n\nIn the case of a DEP setting where target bioparticle (particle) such proteins and cells are in the submicro to nano size, the DEP force applied onto the particles will be extreme small. It is because according to the DEP equation (1), the key influence in particle motion by DEP force was determined by the electric field gradient and the particle volume, which was defined by its radius. Therefore, smaller particle will experience smaller DEP force for particle trapping or separation5. In order to compensate for this unchangeable particle radius variable, the microelectrodes geometry design must be optimized to provide an effective and higher performance of DEP response. To achieve of this, according to the equation (1), one of the optimizations of the microelectrodes geometry design that can be done is to design an electrode that can provide a higher electric field gradient to compensate the small particle radius6. Moreover, the optimization may lead to require lower voltage that can circumvent for joule heating effect, while having high throughput performance for higher particle DEP manipulation ability7.\n\n\n\nFor the case a homogeneous spherical particle in the suspension medium, the general time-averaged DEP force amplitude can be expressed by the classical DEP force as equation (1). εm denoted as the absolute relative permittivity of suspension medium, while ε0= 8.854×10-12 F/mis the permittivity of the vacuum, the spherical particle’s radius is denoted as r or rext,. ERMS is the root-mean-square of the applied electric field in AC, and Re[fCM] is the real part of Clausius-Mossotti (CM) factor2. The equation of the real part of Clausius-Mossotti factor Re[fCM] is given in equation (2).\n\n\n\nThe subscripts “p” and “m” are referred to particle and medium respectively. inline math and inline math are the complex permittivity with respect to particle and medium which can be defined as in equation (3)8\n\n\n\nσp and σm is the electric conductivity of a particle and medium respectively. ω is angular frequency of the applied electric field defined as ω = πƒ, where ƒ is the frequency of the electric field and j=−1.\n\nIn order to optimizing the electrode geometry and design, there are several geometrical parameters that will be affecting the electric field gradient ∇E2 which are the width of the electrodes and the spacing between the two electrodes9,10.\n\nTo further study on this, a rectangular geometry shape electrode has been designed as shown in Figure 1 which is an interdigitated castellated type design which consist of two same-sized electrodes, and these two electrodes are made up of gold material with the thickness of 0.15 µm. The two electrodes were positioned with a spacing gap between them and placed at the bottom of the microfluidic channel, the microelectrode dimension has been shown in Table 1. For 3 main variables with each parameter used to study the optimization of electric field gradient were: 1. the microfluidic channel height (20 µm, 30 µm, 40 µm, 50 µm), 2. the electrode width (20 µm, 40 µm, 60 µm, 80 µm, 100 µm) and 3. the electrode gap spacing (5 µm, 10 µm, 15 µm, 20 µm, 25 µm, 30 µm, 35 µm, 40 µm, 45 µm, 50 µm).\n\n\nSimulation study\n\nA Finite Element Method (FEM) simulation study was performed by using COMSOL Multiphysics version 5.5 software (RRID: RRID:SCR_014767; URL: https://www.comsol.com/comsol-multiphysics). A 2D space dimension FEM simulation analysis has been carried for this study with only considering the cross-section view of the microfluidic change. Electric currents (ec) physics module was added to defines the electrical potential in the fluid domain and the electric field current was governed by the Ohm’s law continuity equation. The simulation study was carried out in the settings of frequency domain (AC). The governing equations for this frequency domain, electric current, electrical conductivity and relative permittivity are expressed as below:\n\n\n\n\n\n\n\n\n\n\n\nJ denoted as electric current density, σ is electrical conductivity, V electrical voltage, E electrical field, D electric displacement field, ε0 is the relative permittivity of air and εr is the relative permittivity of the fluid respected to the air. By solving these equations, the electric field E in the microfluidic system is calculated and the electric field E is in direct relation to the amount of DEP force according to equation (1).\n\nThe cross-section microfluidic and its rectangular geometry electrodes have been created according to the dimension in the Figure 2. Gold material were assigned for both the electrodes with the electrical conductivity, σ = 44.2x106 S/m and relative permittivity, ε = 1, for the medium within the microchannel was a deionized water with its electrical conductivity, σ = 0.0002 S/m and relative permittivity, ε = 78. Under the electric currents (ec) physics module, 10 V peak to peak AC electric potential was set to be applied to the electrodes. The boundaries of one electrode have been input an electric potential V0 = 10V, while the other electrode with its boundaries were input with electric potential V0 = -10 V. The remaining boundaries of the channel were set to be insulated. Extremely fine mesh was generated for this created model. Lastly, a study was generated with electric current model that defines the electrical potential and it is conducted in a frequency domain (AC).\n\nThe numerical results such as electrical potential distribution Vpp, electric field distribution V/m and electric field gradient ∇E2 were generated from this model. This numerical study was performed with 5 different channel height (20µm, 30µm, 40µm, 50µm) and with the input of different combination parameters of electrode width and electrode spacing as according to the Table 2.\n\n\nResults and Discussions\n\nA parametric study on the geometrical parameters of rectangular shape microelectrode for DEP microfluidic platform has been conducted to identify its optimization microfluidic and electrode design for DEP manipulation of micro to nano particles. In this numerical study, an electric field gradient was generated in the model as shown in Figure 3, by further input of different design variables into the study model such channel height, electrode width and electrode spacing and its parameters to study the optimization of the rectangular microelectrode and identify its optimum design parameters. Electric field gradient ∇Ex2 is the key identifier for an effective DEP microfluidic platform, therefore in the generation of the electric field gradient result in this cross-section microfluidic model, only the horizontal component of the electric field gradient ∇Ex2 is considered.\n\nThe result shows the electric field gradient distribution and the electric field contour.\n\nAccording to the 3D plot result from Figure 4 has shown that the electric field gradient ∇Ex2 is exponentially increased when the applied peak-to-peak voltage is increased. However, the electric field gradient remained unchanged to the increase of applied frequency. Although the frequency has not contributed to the electric field gradient but note that it is still an important factor that influence the particle, as the particle DEP response is highly dependent on applied frequency and which is governed by the equation (2) and equation (3)11. This result has shown that the applied voltage has a substantial influence on the electric field gradient, however the applied frequency will have no effect on the electric field gradient. From the generated 3D plot of all the 4 different channel heights as shown in Figure 5, it has been shown in all the graph that the electric field gradient is gradually decreasing when the electrode spacing is increased and the electric field gradient had a drop at certain point when the electrode width increased beyond 40µm and 80µm.\n\nThe 3D plots generated with MATLAB R2019b.\n\nFirstly, for channel height influencing on ∇E2x values are as shown in Figure 6. As observed from the graph, the difference in channel heights (20 µm, 30 µm, 40 µm) have similar pattern and have very little difference on the electrode’s ∇E2x values. The ∇E2x values gradually increased when the electrode spacing increased. Only the channel height of 50µm showed lowest ∇E2x values at the beginning range of electrode spacing from 5 µm to 15 µm. Further to investigate, extend on channel height from 5 µm to 100 µm, with width of electrode 20 µm and electrode spacing 5 µm kept constant as shown in Figure 8. The ∇E2x values have observed a fall between 40 µm to 70 µm channel height. Therefore, the optimum channel height design should be 40 µm.\n\nFor the electrode widths results as shown in Figure 7, when the channel height is 20µm and 30µm, the ∇E2x values for different range of electrode widths are in the same pattern. It has been shown that when the electrode width exceeded 60µm, the ∇E2x values have dwindled about 64% drop in ∇E2x value. For channel height 40µm the drop in ∇E2x values have started early at 40µm electrode width. However, the electrode widths were not much influence on the ∇E2x values when the channel height reached 50µm, but it has been the least amount of ∇E2x values, it has about 63% less ∇E2x values compared to other channel, since then the ∇E2x values were not much fluctuated and constantly stayed within its ∇E2x range. The optimum design for the electrode width is within 60µm and channel height within 30µm.\n\n\nConclusion\n\nBased on the simulation results, the optimum geometrical parameters for rectangular shape electrode design for the strongest DEP force was identified. In this simulation and parametric studies were only focused on the DEP force and did not include other factors such as hydrodynamic forces, joule heating effects, particles Brownian motion in the microfluidic device. As according to this study, 1. The applied voltage has significant influence on electric field gradient, ∇E2x, however the frequency will not have effect on electric field gradient, nonetheless it is strongly responsible to the DEP response of the particle based on the polarizability between the medium and particle, 2. For the optimum design considerations for rectangular shape microelectrode, the electrode width is advised to fall below 60µm and 3. the channel height should below 30µm to obtain the strongest DEP force for particle manipulation in this microfluidic design.", "appendix": "References\n\nKhoshmanesh K, Nahavandi S, Baratchi S, et al.: Dielectrophoretic platforms for bio-microfluidic systems. Biosens Bioelectron. 2011; 26(5): 1800–1814. PubMed Abstract \| Publisher Full Text\n\nZhang H, Chang H, Neuzil P: DEP-on-a-chip: Dielectrophoresis applied to microfluidic platforms. Micromachines (Basel). 2019; 10(6): 423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPethig R: Review article-dielectrophoresis: status of the theory, technology, and applications. Biomicrofluidics. 2010; 4(2): 022811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman NA, Ibrahim F, Yafouz B: Dielectrophoresis for biomedical sciences applications: A review. Sensors (Basel). 2017; 17(3): 449. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaux E, Bier FF, Hölzel R: Electrode-based AC electrokinetics of proteins: A mini-review. Bioelectrochemistry. 2018; 120: 76–82. PubMed Abstract \| Publisher Full Text\n\nHölzel R, Calander N, Chiragwandi Z, et al.: Trapping single molecules by dielectrophoresis. Phys Rev Lett. 2005; 95(12): 128102. PubMed Abstract \| Publisher Full Text\n\nIvory CF, Srivastava SK: Direct current dielectrophoretic simulation of proteins using an array of circular insulating posts. Electrophoresis. 2011; 32(17): 2323–2330. PubMed Abstract \| Publisher Full Text\n\nAl-Ahdal SA, Kayani ABA, Ali MA, et al.: Dielectrophoresis of Amyloid-Beta Proteins as a Microfluidic Template for Alzheimer's Research. Int J Mol Sci. 2019; 20(14): 3595. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeiterer C, Berg S, Eskelinen AP, et al.: Assembling gold nanoparticle chains using an AC electrical field: Electrical detection of organic thiols. Sensors Actuators B Chem. 2013; 176: 368–373. Publisher Full Text\n\nTuukkanen S, Kuzyk A, Toppari JJ, et al.: Trapping of 27 bp – 8 kbp DNA and immobilization of thiol-modified DNA using dielectrophoresis. 2007; 295204. Publisher Full Text\n\nDeivasigamani R, Maidin NNM, Wee MFMR, et al.: Dielectrophoresis prototypic polystyrene particle synchronization toward alive keratinocyte cells for rapid chronic wound healing. Sensors (Basel). 2021; 21(9): 3007. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "128550", "date": "12 Apr 2022", "name": "Reda Abdelbaset", "expertise": [ "Reviewer Expertise I have 8 years of experience in a lab on a chip applications special in Dielectropgoresis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe purpose of this research is to determine the best design for manipulating biological particles by examining several parametric design configuration factors (channel height: 20-50 m; electrode width: 20-100 m; electrode spacing: 5-50 m).\nI mainly have the following concerns:\nIntroduction:\nThe authors should clarify, on what basis did they determine these ranges, they should be related to the type of tested biological particles (i.e. normal cells, cancer cells, stem cells, or bacteria) and the manufacturing capabilities of electrodes (i.e. PCB or CMOS) and microfluidic chamber.\n\nThe suggested configuration of electrodes in Fig.1 is traveling wave dielectrophoresis (TwDEP):\nThe authors should introduce TwDEP.\nThe authors must revise equation 1 because TwDEP mainly depends on the imaginary part of CM.\nSimulation study - The authors should clarify:\nThe reasons for gold material selection.\n\nThe kind of AC waveforms (i.e. sinewave, or square wave)\n\nThe range of the applied frequencies.\nThe authors based their research solely on the electric field, which is insufficient because the COMSOL software also includes a module (particle tracing for fluid flow) that determines whether or not the electric field is capable of performing the required function (manipulation of biological particles) by calculating the dielectrophoretic force on the particles.\nAlso, the authors should study the fluid flow to evaluate the functionality of the channel’s height.\nTherefore this study is insufficient, the authors should add two modules (particle tracing for fluid flow, and creeping flow)\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "208838", "date": "02 Nov 2023", "name": "Mengren Wu", "expertise": [ "Reviewer Expertise Microfluidics", "Dielectrophoresis", "Cell manipulation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 conducted a simulation work to determine the relationship of electric field gradient ∇Ex2 with electrode spacing, width, and channel height. Specifically, they outlined the steps for simulating the DEP operating in the microfluidic channel. In addition, the combination of these parameters has been discussed and concluded in this article. Overall, this article provides a meaningful and clear conclusion for gradient ∇Ex2 in dielectrophoretic microfluidic channels. There are some suggestions for improvement listed:\n\nThere are no bioparticle-related experiments or simulations in this work. It is suggested that authors change the title and remove the bioparticle-related words which is misleading for readers.\n\nFor the FEM simulation study, the description needs to be improved by adding the description of the model used in the simulation experiments such as the laminar flow/ particle trajectory tracing/ AC electric field. Additionally, it is also suggested to give more detailed information about the calculator used in COMSOL such as the time-dependent or static calculator. In the results section, the authors mainly discussed ∇Ex2 with the variable input parameters such as the geometry of the microfluidic channel and microelectrode array. Based on this point, it is recommended that the authors need to involve these keywords in the title.\n\nIf the authors conducted multiple sets of experiments for determining the relationship of electric field gradient with electrode spacing, width, and channel height, it is suggested to include the error analysis and add the error bar on the results diagram.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "199701", "date": "02 Nov 2023", "name": "Adil Mustafa", "expertise": [ "Reviewer Expertise Microfluidics", "Dielectrophoresis and COMSOL simulation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have reported a simulation study covering the effect of various geometrical parameters on the efficiency of DEP-based microfluidic devices. The study itself has merit but needs an overall before submission.\n\nThe authors have plotted the electric field gradient against channel height and electrode width for most of their results. They also need to plot the CM factor for different frequency and electrical conductivity values to see how efficient their design is. They can refer to Ur Rehman et al. (20221) and Mustafa et al. (20222).\n\nThe authors also need to show the feasibility of their design by sorting biological particles using their simulation to increase the reach of their work.\n\nI have general comments about the writing standard which needs considerable improvement. There are a lot of typos and grammatical errors. They need to be corrected before this manuscript can be accepted for indexing. Some of them are as follows:\n\nFigure quality needs to be improved for every figure. Figures 6 and 7 can be combined to have one figure. Can you also explain the initial decrease in electrical gradient between 10-15 um electrode spacing?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "199696", "date": "03 Nov 2023", "name": "Karina Torres-Castro", "expertise": [ "Reviewer Expertise Microfluidic systems coupled with electrokinetics (DEP)", "mechanical separations", "impedance cytometry and acoustofluidics for LOC and OoC applications." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors did a parametric study using Comsol simulations to evaluate the effect of different electrode configurations, and other parameters in electric field intensity spatial resolution (and magnitude) for DEP particle manipulation. This is done to inform the reader on how to design coplanar electrodes to maximize the DEP polarization response for cell or bioparticle manipulation.\n\nIn this article, the authors claim DEP is regarded as \"the most promising\" technique utilized in microfluidic systems. What are the author's criteria to claim is the most promising technique in microfluidics (or add a reference to substantiate this)? In which scenarios is this technique superior to other microfluidic techniques for particle manipulation?\n\nThe authors refer to DelE^2 as the electric field gradient when in fact EdelE or DelE^2 is not the same as the electric field gradient which is just DelE. DelE^2 and DelE have different physical meanings.\n\nDelE^2 (or EDelE) matters and not just the DelE (electric field gradient). EDelE o DelE^2 is the parameter that should be optimized. EDelE will polarize the particles according to their electrical properties, volume, and surrounding medium electric properties.\n\nThis sort of parametric study for optimizing DelE^2 spatial extent using coplanar and 3D electrodes has been reported in previous works (1 and 2).\n\nThere are multiple typos in the text and they use plural verbs/nouns in sentences that are singular. The authors stated that they neglected important forces such as hydrodynamic forces that play a significant role in microfluidic particle manipulation. I encourage them to consider particle drag force and use Comsol's particle tracing module to test DEP's effectiveness in manipulating biologically relevant particles/particles.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-172
https://f1000research.com/articles/9-1249/v1	15 Oct 20	{ "type": "Research Article", "title": "A novel genotyping method to determine copy number in a mouse line commonly used for inducible transgene expression in brain and spinal cord", "authors": [ "Eleanor Hobbs", "Sarah Ryan", "Sara Rollinson", "Stuart Allan", "Stuart Pickering-Brown", "Eleanor Hobbs", "Sara Rollinson", "Stuart Allan", "Stuart Pickering-Brown" ], "abstract": "Background: The NEFH-tTA mouse has the human neurofilament heavy polypeptide promoter directing tetracycline-controlled transactivator protein (tTa) expression to the brain and spinal cord, allowing tissue-specific and doxycycline-suppressible expression of a target gene. Current genotyping protocols can only differentiate between wild-type and transgenic animals. Being able to differentiate between hemizygous and homozygous animals would be beneficial in experiment planning and reducing animal numbers. Methods: We have identified the insertion site of the NEFH-tTA transgene via targeted locus amplification and next-generation sequencing. This was then used to design a multiplex PCR assay to distinguish between hemizygous and homozygous mice. Results: The NEFH-tTA transgene is located on chromosome 12. Our genotyping method can identify hemizygous and homozygous mice. Conclusions: The NEFH-tTA transgenic mouse line is a useful tool for studying a wide range of diseases including frontotemporal dementia and motor neuron disease, as well as other neurodevelopmental, neuromuscular or neurodegenerative disorders. We have designed and utilised a novel genotyping assay to distinguish between hemizygous and homozygous mice, involving a simple PCR assay. This is easily adaptable to a laboratory-specific protocol or machine, and will allow refinement of breeding strategies and a reduction in the number of animals that cannot be used in experiments.", "keywords": [ "NEFH-tTA", "frontotemporal dementia", "FTD", "ALS", "targeted locus amplification", "genotyping", "mouse" ], "content": "Abbreviations\n\nNEFH, neurofilament heavy polypeptide promoter; TG, transgene; tTa, tetracycline-controlled transactivator protein; TRE, tetracycline response element; TLA, targeted locus amplification.\n\n\nIntroduction\n\nThe NEFH-tTA mouse has the human neurofilament heavy polypeptide promoter directing tetracycline-controlled transactivator protein (tTA) expression to large-calibre axons of the brain and spinal cord, allowing tissue-specific and doxycycline-suppressible expression of a target gene1. The line was generated via pronuclear injection of a plasmid with the human NEFH promoter isolated from BAC clone (CHORI: RP11-91J21) to drive expression of the tetracycline transactivator (tTA) gene, which randomly integrated into the genome1. While a genotyping protocol is available to distinguish between wild-type and transgenic animals1, ideally a colony of homozygous animals would be maintained for experimental breeding with another transgenic line with the gene of interest under the control of a tetracycline response element (TRE). All offspring of these breeds would therefore be NEFH-tTA+/- (which has been shown to be a sufficient driver of expression1); this would allow use of littermate controls and reduce required breeding numbers as encouraged by the ARRIVE guidelines2. In order to address this, we have identified the insertion site of the NEFH-tTA transgene, designed a novel PCR genotyping strategy, and demonstrated its reliability in a colony of NEFH-tTA mice.\n\n\nMethods\n\nAll animal work was approved following local ethical review by the University of Manchester Animal Welfare and Ethical Review Board and performed under Home Office project license 70/8903 and in accordance with the Home Office (Animals) Scientific Procedures Act (1986). All efforts were made to ameliorate harm to animals; mice were housed and maintained according to standard practices in the University of Manchester Biological Services Facility (detailed below) and no licenced procedures were performed.\n\nB6;C3-Tg(NEFH-tTA)8Vle/J mice (JAX stock #025397) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and bred with C57BL6/J mice to produce a colony of hemizygous NEFH-tTA animals (13 transgenic mice were crossed with 12 wild-type in total, as this number was sufficient to create a new colony of backcrossed mice). All animal work was carried out in the University of Manchester Biological Services Facility. Mice had free access to food and water and were housed under light-humidity- and temperature-controlled conditions: ambient temperatures of 21°C (±2°C), humidity of 40–50%, 12 h light cycle, ad libitum access to water and standard rodent chow. Animals were housed up to five per cage with environmental enrichment. For breeding, mice were housed as either breeding pairs or trios of two female and one male, and pups housed with parents until weaning (approximately four weeks post-birth).\n\nAt weaning, animals were restrained by scruffing and ear biopsies were taken using a standard ear puncher and genotyped as described1. Briefly, tissue was disrupted in proteinase K for one hour at 57°C; reactions containing 3μL of diluted DNA, 10μL PrimeSTAR HS Premix (Takara, Clontech #R040A) and 0.5μM each of forward and reverse transgene primers and internal positive control primers (Primers 1-4, Table 1) in a total volume of 20μL were amplified in a thermal cycler (ThermoFisher SimpliAmpTM A24812; Table 2). PCR products were visualised on a 2% agarose gel with HyperLadder IV (Bioline) to determine product size.\n\nA combination of targeted locus amplification (TLA) and next-generation sequencing was used to identify the insertion point of the NEFH-tTA transgene. This was performed by Cergentis B.V (Utrecht, The Netherlands). One mouse spleen was dissected from a six-week old NEFH-tTA+/- mouse (culled by Schedule 1 CO2 inhalation) and cells isolated for TLA sample prep. Spleen tissue was disrupted by gently pushing through a 40µm mesh and cells collected in PBS with 10% foetal calf serum (Gibco #10500064). Cells were pelleted by centrifugation at 250g for ten minutes at room temperature, resuspended in lysis buffer (0.15M NH4CL, 0.01M KHCO3, 0.0002M EDTA) and incubated at room temperature for five minutes before additional centrifugation at 250g for ten minutes. Cells were resuspended in PBS with 10% foetal calf serum (Gibco #10500064), stored at -80°C and shipped to Cergentis on dry ice.\n\nTwo primer sets (Table 3) were used in individual TLA amplifications. PCR products were purified and library prepped using the Illumina NexteraXT protocol and sequenced on an Illumina sequencer. Reads were mapped using BWA-SW, which is a Smith-Waterman alignment tool. This allows partial mapping, which is optimally suited for identifying break-spanning reads. The mouse genome version mm10 (GenBank assembly accession: GCA_000001635.2) was used for mapping.\n\nHemizygous mice were bred together (total 25 animals; the number of breeding animals was chosen based on expected number of homozygous offspring and numbers required to set up a large colony to breed with a different transgenic line3) and ear biopsies taken from all offspring at weaning (n=50) as described above. Primers were designed using Primer3 (web version 4.1.0) to produce a product spanning the insertion site on chromosome 12 (Primers 5-6, Table 1) and PCR reactions were set up as described under Maintenance of transgenic mice. REDExtract-N-Amp™ 2X PCR Reaction Mix (Sigma-Aldrich, #R4775) was used instead of PrimeStar.\n\nOutcome vs Expected for genotype was performed using GraphPad Prism version 8.00 for Mac (GraphPad Software, La Jolla California USA, www.graphpad.com).\n\n\nResults\n\nUsing TLA highest coverage is observed on the sequences directly surrounding the location of the primer set. Here, high coverage is observed on chromosome 12, outlined in red in Figure 1A, indicating the transgene (TG) has integrated in chromosome 124.\n\n(A) Targeted locus amplification (TLA) sequence coverage across the mouse genome using primer set 1. The chromosomes are indicated on the y-axis, the chromosomal position on the x-axis. Similar results were obtained with primer set 2 (see Underlying data4). (B) TLA sequence coverage across mouse chromosome 12 with primer pair 1. The arrow points towards the integration site. (C) TLA sequence coverage across the TG with primer pair 1 (top) and 2 (bottom).\n\nFrom locus-wide coverage (Figure 1B) it was concluded that the TG has integrated in mouse chr12:6917896-chr12:6917912. Reads marking the TG integration are identified in Table 4. The 11 bases between chr12:6917896-chr12:6917912 have been deleted following the integration event. According to the reference sequence (mm10) there are no genes annotated in this region. Complete coverage is observed across the whole TG sequence from TG:68-18543 (Figure 1C). At the edges of the coverage, fusion reads have been found, connecting these ends, but the TG: 1 -68 and TG 18544 – end regions are not integrated into the mouse genome (Table 4). These regions contained sequences for bacterial DNA replication and therefore are not required for transgenic expression in the mouse.\n\nHomologous sequences are shown in purple.\n\nNext to the head-to-tail fusion as reported above, four other TG-TG fusions were found within the TG sequence (Table 4). While an exact copy number cannot be determined using TLA, an estimation can be made based on the number of integration sites, number of fusion reads and the ratio of coverage on the TG and genome integration site. Here, one integration site was found and a total of five TG-TG fusions were found. The coverage at the TG was significantly higher compared to the genome. These facts together indicate that >5 copies of the TG have integrated.\n\nWe designed primers to amplify a wild-type PCR product of 488bp from chromosome 12, which would be disrupted by insertion of the transgenic allele (Figure 2A). A multiplex PCR reaction including the original primers to the transgene1 (Table 1) was carried out on samples from a hemizygous intercross and proved able to distinguish between wild-type (488bp band only), hemizygous (both bands) and homozygous (150bp band only) (Figure 2B)4.\n\n(A) Primer design to produce a wild-type product that is disrupted in the presence of the transgenic allele. (B) Samples visualised on a 2% agarose gel showing wild-type (top band), homozygous (bottom band) and hemizygous (both bands) animals.\n\nAfter genotyping 50 offspring of hemizygous intercrosses we observed no significant difference in the number of Nefh-tTa+/+ mice compared to the expected ratio (Figure 3, chi-square=2.64, p<0.26714), confirming our novel genotyping assay performs as expected. Homozygous mice grew to adulthood and did not exhibit any overt phenotype.\n\nThere was no significant difference between expected numbers of homozygous animals and the observed outcome.\n\n\nDiscussion/conclusion\n\nThe NEFH-tTA transgenic mouse line is a useful tool for studying a wide range of diseases including frontotemporal dementia and motor neurone disease, as well as other neurodevelopmental, neuromuscular or neurodegenerative disorders. Here we have shown that multiple copies of the transgene have inserted into chromosome 12. Furthermore, we have designed and utilised a novel genotyping assay to distinguish between hemizygous and homozygous mice, involving a simple PCR assay. This is easily adaptable to a laboratory-specific protocol or machine and will allow researchers using this line to refine their breeding strategies and reduce the number of animals that cannot be used in experiments.\n\n\nData availability\n\nRepository: “A novel genotyping method to determine copy number in a mouse line commonly used for inducible transgene expression in brain and spinal cord – Raw data/analysis” DOI: https://doi.org/10.6084/m9.figshare.12982220.v14.\n\nThis project contains the following underlying data:\n\n- Nefh o vs e.pzfx (raw data and analysis for Figure 3)\n\n- Nefh o vs e.xlsx (raw data and analysis for Figure 3)\n\n- TLA and seq data – Cergentis report 2017UMAN0801.pdf (all sequencing and TLA data received from Cergentis)\n\n- Raw data – PCR gel image.tif (original, unedited PCR gel image from Figure 2)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to thank the University of Manchester Biological Services Facility for animal care.\n\n\nReferences\n\nWalker AK, Spiller KJ, Ge G, et al.: Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta Neuropathol. 2015; 130(5): 643–660. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKilkenny C, Browne WJ, Cuthill IC, et al.: Improving bioscience research reporting: The arrive guidelines for reporting animal research. Animals. 2014; 4(1): 35–44. Publisher Full Text \| Free Full Text\n\nRyan S, Hobbs E, Rollinson S, et al.: CRISPR/Cas9 does not facilitate stable expression of long C9orf72 dipeptides in mice. Neurobiol Aging. 2019; 84: 235.e1-235.e8. PubMed Abstract \| Publisher Full Text\n\nRyan S, Hobbs E, Pickering-Brown S, et al.: Raw data/analysis. figshare. Dataset, 2020. http://www.doi.org/10.6084/m9.figshare.12982220.v1" }	[ { "id": "73226", "date": "29 Oct 2020", "name": "Virginia Lee", "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\nIn this manuscript from Eleanor Hobbs et al., the authors report the use of targeted locus amplification to identify the integration site of the NEFH-tTA transgene in B6;C3-Tg(NEFH-TTA)8Vle/J mice, which have been used in disease models for neuron-specific expression of doxycycline-regulatable cassettes in the brain and spinal cord. Having mapped the NEFH-tTA integration to ch12qA1.1, the authors proceed to develop a simple PCR assay to discriminate mice hemizygous and homozygous for the transgene.\nThe study is well performed and lucidly communicated. The reported PCR assay is anticipated to be of use to investigators breeding B6;C3-Tg(NEFH-TTA)8Vle/J mice, facilitating crossing to generate homozygotes. Some minor amendments would enhance the manuscript and these are discussed below:\nThe reference to ‘copy number’ in the title is misleading since the reported genotyping method actually assesses zygosity of the NEFH-tTA locus rather than NEFH-tTA transgene copy number per se.\n\nThe amplicon reads from targeted locus amplification are aligned to GRCm38 despite an updated assembly - GRCm39 – now being available. While it is unnecessary to repeat the bioinformatic analyses, it would be useful to add the NEFH-tTA 5’ and 3’ integration coordinates in terms of the latest Mm genome build.\n\nIt would be helpful to provide a FASTA reference file for the NEFH-tTA sequence so that readers can more easily interpret the junction and fusion sites identified in Table 4.\n\nThe manuscript indicates that similar targeted locus amplification findings were found for the second primer set and references the online ‘Underlying data’ files (Fig. 1A legend). However, data showing the alignment of NGS reads by chromosome for primer set 2 are not actually in the supplemental files. This would be best resolved by adding the missing data or, less optimally, by removing reference to the second primer pair or clarifying what is meant by “similar results”.\n\nThe molecular biology aspects of the targeted locus amplification method are not described. Moreover, the Illumina instrument used should be identified and the ‘bwa bwasw’ alignment parameters should be given.\n\nThe second paragraph of the Results section indicates that the NEFH-tTA integration coordinates were identified from locus-wide coverage data (Fig. 1B), but presumably, it was actually the sequence of the ch12/TG junction reads that identified the integration site.\n\nThe treatment of data pertaining to the NEFH-tTA integration copy number in the ch12 locus is not entirely clear (results paragraphs 2 and 3). Is it possible that any of the TG-TG fusion amplicons were generated secondary to the fragmentation and re-ligation procedure used in targeted locus amplification? Without orthogonal sequencing of the locus, the reader is left unclear as to the proposed arrangement of the putative multiple transgene copies at the integration site. Left unresolved, the corresponding statement in the conclusion (“…we have shown that multiple copies of the transgene have inserted…”) may be viewed as excessively definitive.\nFigure 3 is superfluous and could be replaced by the Chi-square dependency 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7598", "date": "10 Feb 2022", "name": "Sarah Ryan", "role": "Author Response", "response": "We thank Prof Lee for reviewing the manuscript, and are grateful for the constructive comments. There were some points to be addressed, as follows: The reference to ‘copy number’ in the title is misleading since the reported genotyping method actually assesses zygosity of the NEFH-tTA locus rather than NEFH-tTA transgene copy number per se. We agree this was incorrect use of the phrase “copy number” and have changed the title accordingly. The amplicon reads from targeted locus amplification are aligned to GRCm38 despite an updated assembly - GRCm39 – now being available. While it is unnecessary to repeat the bioinformatic analyses, it would be useful to add the NEFH-tTA 5’ and 3’ integration coordinates in terms of the latest Mm genome build. A BLAST of the integration site sequences using the updated GRCm39 demonstrates that these coordinates are the same as those listed in the manuscript, and therefore no update is necessary in this case. It would be helpful to provide a FASTA reference file for the NEFH-tTA sequence so that readers can more easily interpret the junction and fusion sites identified in Table 4. We thank the reviewer for this useful suggestion and agree this should be included. Since the FASTA is a long text file we have uploaded it as part of the extended/underlying data repository accompanying the article and referred to it in the main text. The manuscript indicates that similar targeted locus amplification findings were found for the second primer set and references the online ‘Underlying data’ files (Fig. 1A legend). However, data showing the alignment of NGS reads by chromosome for primer set 2 are not actually in the supplemental files. This would be best resolved by adding the missing data or, less optimally, by removing reference to the second primer pair or clarifying what is meant by “similar results”. The reason this additional data is not in supplemental files is because F1000 does not allow supplementary data to be added to articles, but rather makes use of the “underlying data” repository to include both raw data and extended data. Therefore the reference to underlying data in this statement should be considered as equivalent to a reference to a supplemental figure in a different journal. The molecular biology aspects of the targeted locus amplification method are not described. Moreover, the Illumina instrument used should be identified and the ‘bwa bwasw’ alignment parameters should be given. Unfortunately we are unable to provide this information as Cergentis performed this part of the analysis using their proprietary methods. However, we have included the full report provided by Cergentis as part of the Underlying Data. The second paragraph of the Results section indicates that the NEFH-tTA integration coordinates were identified from locus-wide coverage data (Fig. 1B), but presumably, it was actually the sequence of the ch12/TG junction reads that identified the integration site. We have edited the text here to further clarify that the locus-wide coverage data allowed us to identify the junction sites and this in turn identified the integration site on chromosome 12. The treatment of data pertaining to the NEFH-tTA integration copy number in the ch12 locus is not entirely clear (results paragraphs 2 and 3). Is it possible that any of the TG-TG fusion amplicons were generated secondary to the fragmentation and re-ligation procedure used in targeted locus amplification? Without orthogonal sequencing of the locus, the reader is left unclear as to the proposed arrangement of the putative multiple transgene copies at the integration site. Left unresolved, the corresponding statement in the conclusion (“…we have shown that multiple copies of the transgene have inserted…”) may be viewed as excessively definitive We have removed this statement from the conclusion, and edited a similar comment in the results section to clarify this is a possibility but not definitive. Figure 3 is superfluous and could be replaced by the Chi-square dependency table. We have removed Figure 3 and replaced with Table 5, as suggested. The GraphPad Prism file containing the full analysis is also available in the Underlying Data repository." } ] }, { "id": "83325", "date": "28 Apr 2021", "name": "Keiichi Ishihara", "expertise": [ "Reviewer Expertise Neuroscience", "Pathological biochemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEleanor Hobbs et al. in this paper, have identified the integrated site of the transgene in the B6;C3-Tg(NEFH-tTA)8Vle/J mice by TLA and also have developed a simple PCR method to distinguish for hemizygous and homozygous. The manuscript is well written, but some typos, infelicities and concerns remain in existence as following:\nAbbreviation: “tTa” correct to “tTA”.\nIntroduction: Definition of “tTA” is duplicated.\nMethods: How many times were the transgenic mice backcrossed to the C57BL/6J mice before they were used in this study?\nResults:\nIn the first paragraph, transgene is abbreviated as TG, but the word, transgene is already appeared in the introduction.\n\nThe figure 3 is better to described as a table, showing number and ratio in each genotype.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7599", "date": "10 Feb 2022", "name": "Sarah Ryan", "role": "Author Response", "response": "We thank Prof Ishihara for reviewing the manuscript, and are grateful for the constructive comments. There were some points to be addressed, as follows: How many times were the transgenic mice backcrossed to the C57BL/6J mice before they were used in this study? Our standard breeding strategy for maintenance of the transgenic line before the genotyping assay was developed was to always cross transgenic mice with wild-type. This was because the pre-existing genotyping assay could only distinguish between transgenic and wild-type but not homo/hemizygous transgenics, and it had previously been reported that homozygous transgenics of this line do not breed well together. Therefore all experimental mice were backcrossed to C57BL/6J several times before use in this study. However, we did not have a specific backcrossing strategy since the transgene is an expression system rather than a particular gene of interest, and we were not investigating any phenotypes in these mice. The figure 3 is better to described as a table, showing number and ratio in each genotype We have removed Figure 3 and replaced with Table 5, as suggested. The GraphPad Prism file containing the full analysis is also available in the Underlying Data repository. Three typing errors were also noted, which we have corrected." } ] }, { "id": "79860", "date": "28 Apr 2021", "name": "Sandy S. Pineda", "expertise": [ "Reviewer Expertise Neuroscience." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript entitled: “A novel genotyping method to determine copy number in a mouse line commonly used for inducible transgene expression in brain and spinal cord”, by Hobbs and colleagues, they present the use of TLA (Targeted locus amplification) analysis to identify the integration site of the NEFH-tTA transgene in B6;C3-Tg(NEFHTTA)8Vle/J mice, and develop a simple genotyping assay to distinguish Heterozygous from Homozygous animals for the transgene. The use of TLA allowed the mapping of the random integration site to ch12qA1.1 in the mouse genome. The study is quite simple, but clear in their objective of providing researchers a new genotyping protocol that would facilitate the handling of the mouse colony and planning of future experiments.\nHowever, there is some minor changes that would improve the manuscript:\nCould the authors include an extended materials and methods sections to include a summary of the TLA method and the bioinformatics commands used? A summary figure, could also be included to summarise the method.\n\nSince TLA was used to get the exact location of the integration, a supplementary FASTA file with the entire sequence including the junctions might help the reader to better understand the insertion site.\n\nI would have suggested to do a high-resolution copy number analysis, as an extra step. I find the use of copy number in the title somewhat misleading, since the only confirmation of the copy number comes indirectly from the TLA analysis.\n\nI do not see the reason for figure 3, it would be best to be removed or to be merged to figure 2. If kept, you will also need to provide more details from the statistical analysis done, maybe adding the table right next to 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? 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": "7600", "date": "10 Feb 2022", "name": "Sarah Ryan", "role": "Author Response", "response": "We thank Dr Pineda for reviewing the manuscript, and are grateful for the constructive comments. There were some points to be addressed, as follows: Could the authors include an extended materials and methods sections to include a summary of the TLA method and the bioinformatics commands used? A summary figure, could also be included to summarise the method. Unfortunately this information is proprietary as a company (Cergentis) performed this analysis for us. However, we have included the full report with all the information provided by Cergentis as part of the Underlying Data repository. Since TLA was used to get the exact location of the integration, a supplementary FASTA file with the entire sequence including the junctions might help the reader to better understand the insertion site. We thank the reviewer for this useful comment and agree the sequence should be included. Since the FASTA is a long text file we have uploaded it as part of the extended/underlying data repository accompanying the article and referred to it in the main text. I would have suggested to do a high-resolution copy number analysis, as an extra step. I find the use of copy number in the title somewhat misleading, since the only confirmation of the copy number comes indirectly from the TLA analysis. We appreciate that our statements regarding copy number were too definitive, and have changed the title to reflect this. We have also removed mention of multiple copies from the conclusion and edited the results section to clarify that multiple copies are a possibility but that we cannot definitively conclude this from this study. I do not see the reason for figure 3, it would be best to be removed or to be merged to figure 2. If kept, you will also need to provide more details from the statistical analysis done, maybe adding the table right next to it. We have removed Figure 3 and replaced with Table 5, as suggested. The GraphPad Prism file containing the full analysis is also available in the Underlying Data repository." } ] } ]	1	https://f1000research.com/articles/9-1249
https://f1000research.com/articles/11-165/v1	10 Feb 22	{ "type": "Research Article", "title": "Effectiveness of Clinical Presentation (CP) Curriculum in teaching clinical medicine to undergraduate medical students: A cross-sectional study.", "authors": [ "Saroj Adhikari Yadav", "Sangeeta Poudel", "Swotantra Gautam", "Sanjay Kumar Jaiswal", "Samikchya Baskota", "Aaradhana Adhikari", "Binod Duwadi", "Nischit Baral", "Sanjay Yadav", "Sangeeta Poudel", "Swotantra Gautam", "Sanjay Kumar Jaiswal", "Samikchya Baskota", "Aaradhana Adhikari", "Binod Duwadi", "Nischit Baral", "Sanjay Yadav" ], "abstract": "Introduction: The Clinical Presentation (CP) curriculum was first formulated in 1990 at the University of Calgary, Canada. Since then, it has been adopted at various medical schools, including Patan Academy of Health Sciences (PAHS), a state-funded medical school in a low-income country (LIC), Nepal. This study aims to evaluate the perceived effectiveness of the CP curriculum by students and faculty at PAHS, and test knowledge retention through a surprise non-routine exam administered to students. Method: This is a cross-sectional study to evaluate the efficacy of the CP curriculum in teaching clinical medicine to the first batch of MBBS students of PAHS School of Medicine. Ethical approval was obtained from the Institutional Review Committee (IRC)-PAHS (Ref no std1505911069). Perceived effectiveness was evaluated using a set of questionnaires for faculty and students. A total of 33 students and 34 faculty filled the perception questionnaires. Subsequently, a questionnaire consisting of 50 Multiple Choice Questions (MCQs) from different clinical medicine disciplines was administered to test students’ knowledge retention. Out of 49 students, 38 participated in the surprise non-routine exam.\n\nResult: A significantly higher number of faculty preferred the CP curriculum compared to the traditional system of teaching clinical medicine (16 vs 11, Kruskal Wallis: 0.023, ie. P-value < 0.05). A significantly higher number of the students liked and recommended CP curriculum in the clinical year of medical education (20 vs. 13 with p-value < 0.05). In the non-routine surprise exam, two thirds of the students scored 60% or above. Conclusion: Both faculty and students perceive that the CP curriculum system is an effective teaching and learning method in medical education, irrespective of their different demographic and positional characteristics. The students’ overall performance was good in surprise, non-routine exams taken without scheduling or reminders.", "keywords": [ "Clinical Presentation Curriculum", "CP Curriculum", "and Medical Education" ], "content": "Introduction\n\nSir William Osler, considered the father of modern medicine, emphasized the teacher's role in helping students to observe and reason. He recommended abolishing the traditional lecture method of instruction.1 Medical education is evolving in response to scientific advances and societal needs.2 A well-organized comprehensive knowledge domain has practical implications in clinical problem solving, and appropriate teaching and learning methods play an important role in achieving the educational goals.3\n\nClinical presentation (CP) is a relatively new and innovative approach to teaching medicine. CP engages medical students in their understanding of the disease process from clinical feature to diagnosis. Students begin studying abnormalities of complaints, examination, and laboratory findings; i.e., signs, symptoms, and laboratory investigations which a patient presents to the doctor with. Students then progress towards diagnosis. The underlying philosophy of the CP Curriculum is that: “The reaction of the human body to an infinite number of insults is always finite and stable over time”.4 For example, if there is any attack on the respiratory system, whether infectious, inflammatory, immunological, traumatic, or iatrogenic; the respiratory system responds through coughing, cyanosis, chest pain, difficulty breathing, noisy breathing, or hemoptysis.4 Thus, the CP Curriculum aims to help students understand the process of moving from “symptoms to diagnosis.”\n\nThe CP curriculum was first formulated in 1990 at the University of Calgary Faculty of Medicine in Canada.5 The curriculum was adopted and redesigned based on local needs at various medical schools worldwide. Patan Academy of Health Sciences (PAHS), a state-funded medical school in Nepal, has adopted several new and innovative approaches in teaching and learning medicine. The CP Curriculum is one of the several approaches adopted by PAHS.6\n\nPAHS medical education team assumes that the CP curriculum is better than traditional lecture-based teaching. In this study we are testing the perceived effectiveness of students and faculty, and the level of knowledge among the students trained by the CP curriculum. The level of knowledge was assessed by marks scored by the students in a surprise non-routine exam without prior information. Perceived effectiveness was based on the thinking/perception of the students and faculty on the effectiveness of the CP curriculum. We assume the CP curriculum is at least not inferior to traditional lecture-based teaching.\n\n\nMethods\n\nThis is a cross-sectional study that aims to evaluate the efficacy of the CP curriculum in teaching different disciplines of clinical medicine to undergraduate medical students of PAHS, which is currently the only medical school implementing the CP-curriculum in undergraduate medical education. A new Multiple-Choice Question (MCQ) based questionnaire was designed to evaluate the level of knowledge and two separate questionnaires were developed for faculty to evaluate perception about CP-curriculum.\n\nAll consenting medical students from 2016 of PAHS School of Medicine currently in clinical clerkship years and all clinical sciences faculty who had delivered at least one teaching-session with the CP curriculum to these students were included in the study. Consenting students were asked to fill the questionnaire together in class, whereas faculty were approached personally and asked to complete the questionnaires. Students and faculty who were part of the study team, those who didn’t provide consent, and those who participated in the pilot survey section of the questionnaire developed for this study were excluded. All 34 faculty completed the perception questionnaires, with zero non-response rate. Out of 49 students, 33 completed the perception questionnaires and 38 turned up to the surprise non-routine exam for assessment of knowledge retention.\n\nThis study was approved by the Institutional Review Committee (Ethical Committee) of Patan Academy of Health Sciences (PAHS), Kathmandu, Nepal (Ref No std1505911069). Written informed consent was obtained from all participants before completing the questionnaire. Students who gave verbal consent were asked to complete the questionnaire together in class. Faculty were approached personally and requested to complete the questionnaires. At the start of each questionnaire, a tick box was used for participants to indicate written consent. Participants were informed verbally and in writing that their names and identifiying information would be kept anonymous, and their data would only be used for research purposes.\n\nThree sets of questionnaires were used. The first set of questionnaires were designed to test the perceived effectiveness of the CP curriculum from the faculty perspective. It contained seven questions on background information (age, sex, job position, highest academic degree, etc) and 13 questions on perceived effectiveness.\n\nSimilarly, the second set of questionnaires for the students included 11 questions on background information and 15 questions on perceived effectiveness. The perception questionnaire had questions about effectiveness or satisfaction in regard to different aspects of the CP curriculum. Participants had to respond with a tick mark in a Likert scale ranging from one (strongly agree) to five (strongly disagree) for each question.\n\nThe third set of questionnaires tested the students' clinical knowledge and contained 50 MCQs from different clinical medicine disciplines. Based on curriculum of the university, there were seven MCQs each from surgery, medicine, pediatrics, obstetrics and gynecology, and two questions each from orthopedics, emergency medicine, general practice, otolaryngology, anesthesiology, dermatology, dentistry, psychiatry, radiology, ophthalmology, and forensic medicine. The questions were randomly selected from the question pool of the Examination section of university. The selected questions were randomly arranged, and a surprise non-routine written exam was conducted with this questionnaire. A maximum time of one hour was provided to solve these 50 questions.\n\nThese questionnaires were compiled and discussed in the research group and reviewed by the research advisors to establish content validity. Copies of all three questionnaires can be found under Extended data.10 They were administered to randomly selected 15 students and 15 faculty in a pilot study to establish the face validity and feasibility. The students and faculty randomly selected for the pilot study were administered the questionnaires to complete. Then they were asked in detail about the questionnaire and any suggestions for revisions or editing needed. The pilot survey was not powered for statistical comparisons. Only a few grammatical corrections were made after review and feedback from the pilot study. Subsequently, the final study was conducted.\n\nThe faculty participants were also involved in the development of the CP curriculum at PAHS, hence, responder bias in favor of CP curriculum may be present in this study.\n\nThe data collected were digitalized using Epi-Info version 7 software. These raw data were exported to MS-Excel. The excel sheet is made available in the public domain for readers.10 SPSS version 13.0 was used for statistical test and analysis. Shapiro-Wilk test was used first to test the normality. Non-parametric tests (Mann-Whitney and Kruskal Wallis) was used for normal distribution. Classical ANOVA for equal variance and Welch ANOVA for unequal variance were used after testing the homogeneity of variance, and post-hoc/tukey test was used for significant classical ANOVA results.\n\n\nResults\n\nIn this study, we calculated the total score via forced Likert scale, ranging from 1 (strongly agree) to 5 (strongly disagree) for each respondent determined as the dependent variable, and compared it with other variables i.e., background information. The total score of all the Likert scale questions was calculated, and the normality test was performed, keeping “total score” as the dependent variable. The full dataset can be found under Underlying data.10\n\nThe data of the total score did not follow a normal distribution (Shapiro-Wilk Test, p < 0.05), so a non-parametric test was used to compare the dependent variable. We used Mann-Whitney and Kruskal Wallis tests for the variables containing two groups and more than two groups, respectively.\n\nAmong the 34 respondents from the faculty group, 24 (70.59%) were male, and 10 (29.41%) were female. 20 (58.82%) of the faculty respondents were lecturers, and the remaining 14 (41.18%) were senior professors, associate professors, and assistant professors. Out of the 34 respondents, 31 (91.18%) were involved in developing the CP curriculum at PAHS. However, 3 (8.82%) were involved in teaching the curriculum but not in developing the CP curriculum.\n\nAs many as 15 (44.12%) respondents favored the CP curriculum system over the traditional system, 11 (32.35%) preferred the traditional teaching system, and 8 (23.53%) preferred both. Overall, the faculty liked the CP curriculum more than the traditional system of teaching clinical medicine (Kruskal Wallis = 0.023, p-value < 0.05). The majority of faculty, 27 (79.41%), would suggest future students to join a medical school that implemented the CP curriculum system rather than the traditional system. Only 12 (35.29%) of them thought that the CP curriculum system should be the sole leading teaching and learning system in clinical medicine, meaning more faculty preferred a hybrid system of both the CP curriculum and the traditional system. However, these differences were not statistically significant (p-value > 0.05).\n\nAs shown in Table 1, a significant number of faculty (p values > 0.05) perceive the CP curriculum to be more effective than the traditional system for teaching clinical medicine to undergraduate medical students. There is no significant difference in the perception of the effectiveness of the CP curriculum among faculty based on academic rank, gender, highest academic degree, or the institution of their residency training (p-value > 0.05). The median number of faculty who perceive the CP curriculum system to be more effective and suggest future students to study medicine in this system rather than the traditional system is higher. But, the difference was not statistically significant (p > 0.05). There was no significant difference in faculty foreseeing the CP curriculum as the leading method of teaching and learning medical education in the future (p > 0.05).\n\nThe normality test shows that the total score data follows a normal distribution (Shapiro-Wilk, p > 0.05) with a mean value of 50.57 with a standard deviation of 8.17. Therefore, we used a parametric test to compare the test variable with others. We subsequently tested for homogeneity of variance: we used classical ANOVA for equal variance, and Welch ANOVA for unequal variance. Finally, if significant classical ANOVA results were obtained, we used the post-hoc/tukey test.\n\nThere were 33 respondents, among which 23 (69.70%) were males, and 10 (30.30%) were females. The age group of respondents was between 20 to 30 years. A significantly higher number (20 i.e., 60.61%) of the respondents recommended studying in a medical school implementing CP curriculum (p < 0.05). No significant differences were seen between educational or geographical backgrounds and scholarship categories (p > 0.05) as shown in Table 2.\n\nAn hourly surprise non-routine written exam was conducted to test the knowledge of the students. A copy of this exam can be found under Extended data.10 The exam included 50 MCQs from different disciplines of clinical medicine. The surprise test was conducted without prior reminders, and 38 out of 49 students participated. The findings, as outlined in Table 3, show that 24 out of 38 (65.79%) of the students scored 60% or higher. The results demonstrate that approximately two-thirds of the students passed the surprise test, indicating good test performance.\n\n\nDiscussion\n\nThe current study shows a higher preference for the CP curriculum by undergraduate medical students and faculty at PAHS for teaching and learning clinical medicine in medical school. These findings further substantiate previous reviews on the principles of teaching methods and the acceptability of the curriculum.\n\nThis curriculum was chosen in part because of confidence in the comprehensiveness of the knowledge it encompasses. Equally important was the organization of medical knowledge that this curriculum engenders: each clinical presentation is organized according to a variable number of causal diagnostic categories. Each of these categories is identified by a prototype. Exhaustive lists of diagnoses belonging to a given category are avoided. As students' clinical experiences increase and they encounter more diagnoses, the students can add them to the appropriate causal categories stored in their memories. How the diagnostic prototypes are presented allows students to identify the discriminating features within and between each. The process by which students can compare and contrast the distinctive features of each disease is facilitated. It is so because the CP curriculum is well organized and comprehensive.3,7 Since the CP curriculum is simple to follow and to organize the learning content, students in the CP curriculum also reported less stress due to the volume and complexity of study materials and examinations.7\n\nPrior studies at the University of Calgary demonstrated a substantial effect size on students’ retention of basic science knowledge while participating in the CP curriculum.8 Our study conducted on clinical clerkship year participants showed that two-thirds of students achieved 60% (passing scores) or more in the surprise non-routine exam, signifying a high retention of clinical discipline knowledge. Findings from the current study expand on the effectiveness of the curriculum across medical school years with respect to knowledge retention.\n\nA study done among medical students utilizing the CP curriculum showed a favorable response to the use of schema in the CP curriculum.9 In our study, we could not evaluate the use the schemas of CP to perform clinical assessment in order to reach the appropriate diagnosis. We recommend further studies in this respect. Additionally, long-term knowledge retention was not tested in our study, which could be another important area of investigation.\n\nThis study has several other limitations as well. The study was conducted at a single institution, thereby potentially reducing the overall generalizability of the findings. The faculty members recruited as participants for assessing the perceived effectiveness of the curriculum were also involved in the adaptation and development of curriculum at PAHS, hence, potentially increasing responder’s bias in the study by some degree. The cross-sectional nature of the study provides only a limited understanding of the effects of the curriculum over the long term.\n\n\nConclusion\n\nBased on this study, we can conclude that both faculty and students perceive the CP curriculum system as an effective teaching and learning method in medicine, irrespective of their demographic and positional characteristics. The findings suggest higher knowledge retention in knowledge by implementing the CP curriculum during clinical clerkship years. Since the 1990s, CP Curriculum has been established as a multidimensional teaching-learning method in many medical school systems. In the evolving medical education world with rapid digitization, massive turnover of medical and education data, and increased use of remote learning methods, a deeper understanding of influencing variables will help effectively utilize this highly valued curriculum.\n\n\nData availability\n\nFigshare: CP Curriculum Raw data updated in Excel and PDF. https://doi.org/10.6084/m9.figshare.18666410.v110\n\nThis project contains the following underlying data:\n\n- Analysis and Raw data.xlsx\n\nThis project contains the following extended data:\n\n- CP Questionnaire for Faculties.pdf\n\n- CP Questionnaire for students.pdf\n\n- CP Surprise exam Questionnaire.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthors' contributions\n\nSAY, SKJ, AA, and BD conceptualized and designed the study. All 9 authors; SAY, SJ, SP, SG, SB, AA, BD, NB, and SY contributed to data analysis and interpretation. SAY and SP wrote the first draft of the article. All 9 authors, SAY, SP, SG, SJ, SB, AA, BD, NB, and SY critically revised the manuscript and approved the final version of manuscript for publication.", "appendix": "Acknowledgements\n\nWe thank Prof. Dr. Kedar Prasad Baral and Prof. (Associate) Shital Bhandary for their immense help during this research. We thank all respondent faculty and medical students of PAHS for participating in the study.\n\n\nReferences\n\nOsler W: An introductory address on examinations, examiners, and examinees. Lancet. 1913; 182: 1047–1050. Publisher Full Text\n\nBuja LM: Medical education today: all that glitters is not gold. BMC Med. Educ. 2019; 19(1): 110. PubMed Abstract \| Publisher Full Text\n\nMandin H, Jones A, Woloschuk W, et al.: Helping students learn to think like experts when solving clinical problems. Acad. Med. 1997; 72(3): 173–179. PubMed Abstract \| Publisher Full Text\n\nCurriculum for MBBS program of PAHS 2010: Accessed from pahs.edu.np. Full text: Reference Source\n\nMandin H, Harasym P, Eagle C, et al.: Developing a “clinical presentation” curriculum at the University of Calgary. Acad. Med. 1995 Mar; 70(3): 186–193. PubMed Abstract \| Publisher Full Text\n\nShrestha S: Development and implementation of clinical presentation curriculum at PAHS School of Medicine. Journal of Patan Academy of Health Sciences. 2020 Aug; 7(2): 124–128. Publisher Full Text\n\nWoloschuk W, Harasym P, Mandin H: Implementing a Clinical Presentation Curriculum: Impact on Student Stress and Workload. Teach. Learn. Med. 1998; 10(1): 44–50. Publisher Full Text\n\nWoloschuk W, Mandin H, Harasym P, et al.: Retention of basic science knowledge: a comparison between body system-based and clinical presentation curricula. Teach. Learn. Med. 2004 Spring; 16(2): 116–122. PubMed Abstract \| Publisher Full Text\n\nWoloschuk W, Harasym P, Mandin H, et al.: Use of scheme-based problem solving: an evaluation of the implementation and utilization of schemes in a clinical presentation curriculum. Med. Educ. 2000; 34: 437–442. PubMed Abstract \| Publisher Full Text\n\nAdhikari Yadav S: CP Curriculum Raw data updated in Excel and PDF. figshare. Dataset. 2022. Publisher Full Text" }	[ { "id": "123301", "date": "17 Feb 2022", "name": "Priyanka Panday", "expertise": [ "Reviewer Expertise Endocrine disorders", "Heart conditions", "Medications", "COVID-19", "Obstetric conditions", "Epilepsy", "HIV", "etc." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 focuses on the importance of the clinical presentation (CP) curriculum in a particular institute (Patan Academy of Health Sciences (PAHS)) among medical students and faculty in terms of their preference and performance on a surprise non-routine exam.\n\nCross-sectional study is appropriate as a study design for this article.\n\nRelevant articles from 2020, 2019, and 2004 have been appropriately cited as references.\n\nThe methods used for data collection, as well as the result of the study has been elaborated in detail to ensure accuracy.\n\nResults are presented in a tabular form and the conclusion derived coincides with the results indicating the effectiveness of the CP curriculum system as an effective teaching and learning method in medicine.\n\nAs far as the statistical analysis is concerned, it is not my area of expertise. However, p< 0.05 for response of effective implementation of the CP curriculum and the response from faculty is statistically significant.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "123304", "date": "18 Mar 2022", "name": "Jayadevan Sreedharan", "expertise": [ "Reviewer Expertise Epidemiology", "Biostatistics", "Medical education", "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\nTitle: Effectiveness of Clinical Presentation (CP) Curriculum in teaching clinical medicine to undergraduate medical students: A cross-sectional study.\nThis study aimed to assess the perceived effectiveness of students and faculty and the level of knowledge among the students trained by the CP curriculum. The authors assume the CP curriculum is not inferior to traditional lecture-based teaching.\nAre sufficient details of methods and analysis provided to allow replication by others?: It is not clear why the authors have given MCQ to the faculty (their score is given and statistical test done).\nIf applicable, is the statistical analysis and its interpretation appropriate?: The authors mentioned in the article that \"Classical ANOVA for equal variance and Welch ANOVA for unequal variance were used after testing the homogeneity of variance, and post-hoc/Tukey test was used for significant classical ANOVA results\", where they have used this test is not clear in the manuscript.\nThe p-value is given in exact value; the importance of p-value is to check whether to accept the null or alternate hypothesis. In the methodology, they mentioned that p-value >0.05 is not significant. Then what more information do the readers get if they include the actual p-value?\n\nThe sample size of this study is very small and the conclusion from this study can not be generalised to the entire population.\n\nAre the conclusions drawn adequately supported by the results? The authors mentioned in the conclusion that \"The findings suggest higher knowledge retention in knowledge by implementing the CP curriculum during clinical clerkship years\". How the authors reach this conclusion is unclear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": [] }, { "id": "123303", "date": "29 Mar 2022", "name": "Richa Shah", "expertise": [ "Reviewer Expertise Global health", "gerontology", "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\nDear author(s), Thank you for your hard work on this research manuscript. Please find my comments/ queries below.\nThe research article deals with the effectiveness of Clinical Presentation (CP) curriculum in teaching clinical medicine to undergraduate medical students. CP curriculum is yet to be adopted in many low- and middle-income countries. The results show that the medical students and the faculty were satisfied with the CP curriculum and believed CP as a stand-alone method of teaching as well as in conjunction with traditional methods of teaching could benefit medical students.\nStudy design: “This is a cross-sectional study that aims to evaluate the efficacy of the CP curriculum in teaching different disciplines of clinical medicine to undergraduate medical students of PAHS, which is currently the only medical school implementing the CP-curriculum in undergraduate medical education.”\nIs PAHS the only medical school implementing the CP-curriculum in Nepal or worldwide?\n\nEthics and consent: “Students who gave verbal consent were asked to complete the questionnaire together in class.”\nPlease elaborate on this sentence.\n\nWas any faculty member present in the class?\n\nWas the test compulsory or optional?\n\nDid the students have the right to refuse the test or leave the test in between?\n\nData collection:\nWas the questionnaire in English?\n\nHow long did the questionnaire take to complete?\n\nHow much time were the respondents given to complete the questionnaire?\n\nAnalysis:\nWhat were the minimum and maximum possible scores (total or based on questionnaire sets)?\n\nI hope the comments are useful and would enable the author(s) to strengthen this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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/11-165
https://f1000research.com/articles/10-1288/v1	15 Dec 21	{ "type": "Review", "title": "Contribution of BCR-ABL molecular variants and leukemic stem cells in response and resistance to tyrosine kinase inhibitors: a review", "authors": [ "Mohammad Al Hamad" ], "abstract": "Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm generated by reciprocal chromosomal translocation, t (9; 22) (q34; q11) in the transformed hematopoietic stem cell. Tyrosine kinase inhibitors (TKIs) target the mature proliferating BCR-ABL cells, the major CML driver, and increase overall and disease-free survival. However, mutant clones, pre-existing or due to therapy, develop resistance against TKIs. BCR-ABL1 oncoprotein activates various molecular pathways including the RAS/RAF/MEK/ERK pathway, JAK2/STAT pathway, and PI3K/AKT/mTOR pathway. Stimulation of these pathways in TKI resistant CML patients, make them a new target. Moreover, a small proportion of CML cells, leukemic stem cells (LSCs), persist during the TKI therapy and sustain the disease in the patient. Engraftment of LSCs in the bone marrow niche and dysregulation of miRNA participate greatly in the TKI resistance. Current efforts are needed for determining the reason behind TKI resistance, identification, and elimination of CML LSC might be of great need for cancer cure.", "keywords": [ "Chronic myeloid leukemia", "BCR-ABL", "Kinase domain mutations", "Leukemic stem cell", "Tyrosine kinase inhibitors", "mi RNA." ], "content": "Introduction\n\nThe BCR-ABL gene results from a reciprocal translocation t (9; 22) (q34; q11) in the transformed hematopoietic stem cell (HSC).1–3 BCR-ABL is considered the most common genetic abnormality in chronic myeloid leukemia (CML) (95%), followed by acute lymphocytic leukemia (ALL) (35%), and rarely presented in acute myelogenous leukemia (AML) (1%).4–6 The most detected variant of BCR-ABL in CML is P210 BCR-ABL1 that occasionally presents in ALL and AML. The P210 BCR-ABL1 variant expresses in about 48% of Ph+ CML cases, while 52% co-express P210 BCR-ABL1 and P190 BCR-ABL1 variants.7 Approximately 50% of CML patients present without symptoms and are diagnosed incidentally after routine laboratory tests.8 Cytogenetics is the standard diagnostic tool of CML; the Ph chromosome is detected in 90% of the cases. However, 5% of CML cases have a cryptic Ph chromosome that could not be detected by karyotyping. The cryptic Ph chromosome can be detected by fluorescence in situ hybridization (FISH) and/or real-time polymerase chain reaction (PCR).9\n\nClinically CML is divided into three different stages: chronic phase (CP), accelerated phase (AP), and blast phase (BC). Without intervention treatment, the disease progresses from the chronic phase to blast phase which is most likely accompanied by multiple genomic abnormalities. Here we present a review of the BCR-ABL response and resistance to TKI therapy.\n\n\nBCR-ABL gene & protein\n\nBCR-ABL results in the juxtaposition of 3′ sequences of ABL (Abelson proto-oncogene) on chromosome 9 and the 5′ sequences of the truncated BCR (breakpoint cluster region) on chromosome 22. Consequently, the BCR/ABL fusion gene forms on chromosome 22 that encodes BCR-ABL1 oncoprotein, a constitutively active tyrosine kinase.10–12\n\nThe constitutive tyrosine kinase activity of the chimeric BCR-ABL1 oncoprotein, deregulates the downstream signaling of many pathways which results in uncontrolled proliferation, arresting the differentiation of the HSC, and limits apoptosis. Consequently, the normal HSC transforms into leukemic stem cells (LSC), which accumulates in the bone marrow (BM) and thus replaces the normal stem cells.13,14\n\nThree different BCR-ABL chimeric proteins emerge from the fusion of mRNA molecules of different lengths of the ABL gene with the BCR gene. The most common breakpoint in BCR occurs in intron 13 or intron 14, then exon 13, or 14 exons (M-BCR) fused to the ABL1 gene at exon a2, which is referred to as e13a2, e14a2. These fusions result in a BCR-ABL1 protein with 210 kilodaltons molecular mass (p210 BCR-ABL1), present in 95% of CML. The second alternative (<1%) is that the e19a2 fusion transcript produces a larger fusion protein with 230 kilodalton weight (p230 BCR-ABL1), a diagnostic marker for neutrophilic-chronic myeloid leukemia. The third alternative is p190 BCR-ABL1 protein resulting from e1a2 fusion transcript,6 most common in B cell ALL, less so in AML, and rarely presented in CML.\n\nBCR-ABL oncoprotein possesses different domains from BCR and ABL. The BCR portion includes the NH2-terminal coil-coil (CC) domain (amino acid 1-63), an oligomerization domain, followed by a serine/threonine kinase domain-containing Tyr 177, a binding site for growth factor receptor-binding protein 2 (GRB2), and Ras homolog gene/guanine nucleotide exchange factor (Rho/GEF) kinase domain, a GTPase activating protein that gets activated when binded to GTP and turned off when binded to GDP, and thus control the activation of Ras protein. Whereas ABL domains include Src- homology (SH) SH3, SH2, and SH1 (Kinase) domains, followed by proline-rich domain and binding domain (BD), and facilitate the nuclear protein, actin, and DNA binding. CC domain and Y177 are required for the efficient activation of ABL kinase. Tetramerization of BCR-ABL is essential for constitutive kinase activity. Targeting the CC domain deregulates the tetramerization of BCR-ABL, reduces the kinase activity, and increases sensitivity to imatinib (tyrosine kinase inhibitor).15,16\n\nIn BCR-ABL, Y177 plays a crucial role in leukemic progenitor cell expansion, proliferation, and survival through activation of the Ras and PI3K/Akt pathway. Interestingly, mutated 177Y fail to develop CML and increase sensitivity to imatinib, suggesting a target of Y177 to eliminate the leukemic stem cells.17 Rho/GEF retained in p230 and p210 BCR-ABL but not in p190, thought to facilitate calcium-dependent lipid binding, restrict proliferation and support survival.18\n\nABL protein has a tyrosine kinase function that is regulated by SH2 and SH3 domains in the N-terminal end. SH3 is considered as a negative regulator of the tyrosine kinase activity, therefore inhibition of SH3 leads to an abnormal increase in the enzyme activity and enhances the malignant transformation.19\n\nThe SH1 domain of ABL has an autophosphorylation site that activates kinase as conjugates to ATP. However, ATP-binding inhibitors, compete with ATP on its binding site and impeding the downstream intracellular signaling pathway.20,21 SH2, the protein-protein interaction domain, activates the SH1 by establishing a tight junction with the N-terminal lobe of SH1, resulting in downstream activation of the intracellular signaling pathways. Interestingly, the open conformation of SH1 allows interacting with the SH2 domain, whereas the close conformation prevents this binding. The SH3 of ABL binds to SH1 through N-terminal myristoyl modification, which induces conformational changes on SH1 that allow SH2-SH3 docking onto it.22\n\nIn a normal physiological state, tyrosine kinase (TK) regulates the activities of the cell cycle. Constitutive activation of TK interrupts the normal state of the cell cycle, resulting in inhibition of cell differentiation, uninterrupted activation of the cell proliferation, and escapes cell apoptosis. Thus, BCR-ABL deregulates many pathways.23\n\nABL protein shuttles between the nucleus and cytoplasm; however, the chimeric BCR-ABL oncoprotein stops this shuttle and retains ABL protein in the cytoplasm, where it interacts with many different proteins that are involved in the malignant transformation.24\n\nIn BCR-ABL fusion, the N-terminal oligomerization domain of BCR activates the ABL tyrosine kinase by blocking the SH3 on the N-terminal of ABL1.25 Accordingly, BCR-ABL oncoprotein activates diverse signaling pathways including the RAS pathway, JAK2/STAT pathway, and PI3K-AKT-mTOR pathway. BCR-ABL oncoprotein directly activates JAK2/STAT pathway and continuously enhances cell survival in CML.26\n\n\nResponse and resistance to TKI\n\nTKIs have dramatically changed the treatment and prognosis of CML. TKI targets the proliferating mature BCR-ABL cells which are considered the major driver of CML. However, a minority of subclones with genetic aberration and quiescent leukemic stem cells are not eliminated by TKI.27,28 Molecular resistance is measured by quantitative real-time polymerase chain reaction (qRT-PCR) of BCR-ABL1 and cytogenetic resistance (Ph+ persistence). These procedures are considered the main standard for monitoring the patient response to therapy, predicting relapse, and guide treatment decisions.29\n\nBinding of ATP to tyrosine kinase active site of BCR-ABL oncoprotein phosphorylate tyrosine residue of substrate results in progression of CML. TKI impedes the ATP- tyrosine kinase binding and thereby halts the constitutive tyrosine kinase activity of the BCR-ABL1 oncoprotein and thus inhibits the CML progression. There are three types of TKI inhibitors. Type I is ATP competitive inhibitors like imatinib and nilotinib which compete with ATP in the ATP binding site of the kinase domain. This, consequently, prevents autophosphorylation, substrate phosphorylation and thus inhibits proliferation and induces apoptosis for BCR-ABL cells. Type II is ATP competitive inhibitors like dasatinib, which bind to the ATP-binding site as well as the hydrophobic adjacent binding site which is accessible only when the kinase is in an inactive configuration.30 The third type is allosteric inhibition which involves the binding of TKI to the allosteric-myristate binding pocket, induces a conformational change in the kinase domain and renders the kinase-inactive.31 It has been estimated that about 25% of CML patients develop resistance to TKI that necessitates to switch TKIs at least once during their lifetime.32\n\nResistance to imatinib occurs in approximately 17% of patients with 5 years of follow-up. The mechanism of TKI resistance is subdivided into ABL1 mutation-dependent (acquired resistance) which means loss of response, and ABL1 mutation-independent (primary resistance), which results in conformational changes of the TKI binding site due to mutation in SH1 of ABL1.33 Therefore, a new target therapy or combining TKI is required to cure CML. As a result, the new version of TKIs, dasatinib, and bosutinib, have been approved as first-line therapy for imatinib resistance patients.34\n\nThe dominant mechanism of imatinib resistance is acquired mutation in the kinase domain (KD) of BCR-ABL with greater frequency in AP, BP CML patients than CP.35,36 These mutations reside in phosphate binding loop at positions M244, G250, Q252H, Y253H/F, and E255K/V, gatekeeper (T315, F317), the activation lobe (H396), SH2, and C-lobe (M351, F359) that ultimately impaired TKI binding by affecting the essential residue for direct contact or by preventing the inactive conformation of ABL kinase domain appropriate for imatinib binding37 (Figure 1). Nilotinib shares the same binding site of imatinib with significantly higher affinity that overcomes many imatinib-resistant mutations except for Y253, E255, T315I, and F359 mutations.38,39 About 25% of CML patients develop T315I mutation that inhibits TKI binding and develop resistance in all TKIs except ponatinib.40 T315I mutation, which occurs in gatekeeper residue of the ABL domain, affects the binding affinity of imatinib, dasatinib, bosutinib, and nilotinib to the ATP-binding site.34,38 Mutations within the ABL kinase domain and overexpression SRC are among the imatinib resistance mechanisms in CML. Dasatinib and bosutinib have dual inhibitory action against SRC and ABL kinase that address most of the BCR-ABL mutations associated with TKI resistance except T315I.41,42 Ponatinib, a pan-BCR-ABL inhibitor, has a great efficacy against native and mutated BCR-ABL1 including the T315I variant.43 However, a dose-dependent increased risk of developing arterial occlusion, stroke, limb ischemia, and other vascular events has been found.44,45 Asciminib is an allosteric inhibitor, unlike other TKI inhibitors, that binds the myristoyl pocket of BCR-ABL. Asciminib is a potent and highly selective inhibitor of both native and mutated BCR-ABL including the gatekeeper T315I mutant. Asciminib is considered a relatively safe option for CP or AP CML with resistance to the currently available TKIs46,47 (Figure 2).\n\nBCR-ABL-independent resistance might develop through upregulation of the PI3K/AKT/mTOR pathway.48 At this point, PI3K/AKT/mTOR pathway inhibitors might be a new target for TKI resistance in CML patients.49 NVP-BEZ235 is a dual inhibitor which shows efficacy against imatinib-resistance CML by arresting the cell cycle at G0/G1 and inactivating the PI3K/AKT/mTOR pathway.50\n\n\nCML leukemic stem cells\n\nCML leukemia stem cells (LSCs) are subpopulations of CML cells that persist through TKI therapy and sustain the disease in patients. LSCs are quiescent cells that reside in the microenvironment of BM and remain a potential reservoir for disease relapse. LSCs are characterized by high heterogeneity of genetic, epigenetic, and transcriptional mechanisms.51 The persistent LSCs result from resistance to targeted therapy that might happen primarily as a lack of initial response to treatment, or acquired as disease relapses after initial response to the therapy.52 Of crucial importance, determination, and elimination of LSC might be of great need for cancer cure.\n\nCD34+/CD38- is a stem cell marker of both normal stem cells and LSC.53 CD34+/CD38- LSC in CML patients was found to express higher levels of CD33 compared to normal CD34+/CD38- stem cells.54 Recently, Herrmann et al. demonstrated that CD34+/CD38- CML-LSC express higher levels of CD 25, CD33, and CD 125 compared to normal BM stem cells. Moreover, immunological targeted therapy against these markers resulted in depletion of CML-LSC and reduce LSC engraftment.55 In a study of single-cell gene expression analysis combined with an immunophenotypic screening of CML-LSC, an aberrant expression of CML-LSC surface markers CD25, CD26, and interleukin1 receptor accessory protein (IL1RAP) was detected. These markers were found to be downregulated after TKI administration.52 In contrast to normal stem cells, IL1RAP is a tight marker of BCR-ABL positive cells, and CD176 is a hematopoietic stem cell marker, co-expressed on CD34+/BCR-ABL+ cells of peripheral blood from CML patients. Interestingly, a bivalent antibody specific to both IL1PRAP and CD176 was found to target the cell population of CML but not their corresponding normal cells.56 Another study demonstrated that BCR-ABL CML CD34+/CD38-/CD26+LSC cells are resistant to TKI.57 Interestingly, it turned out that the pathways of TGF-ß and TNF-α were dysregulated in both BCR-ABL- and BCR-ABL+ CML-LSC with increased LSC quiescence and resistance to TKI. Moreover, increased serum levels of TGF-ß and TNF-α at diagnosis were associated with low treatment response in CML that might have an important role as a clinical predictive biomarker.52 Codavarthy et al. demonstrated that expression of CD44 on BCR-ABL + LSC and E-selectin on bone marrow endothelium participates greatly in engraftment of LSC in bone marrow niche which protects the LSC from imatinib treatment. Interestingly, GMI-1271, an E-selectin inhibitor, showed that releasing of BCR-ABL+ from the bone marrow niche increase responsiveness to Imatinib by decreasing the leukocyte counts and BCR-ABL cells.58 Bone morphogenetic proteins (BMP) are soluble growth factors that regulate stem cell fate and proliferation. The concentration of BMP2 and BMP4 was found to be higher in the bone marrow of CML patients in CP corresponding to normal bone marrow donors.59 BMP bind to its receptor, BMP receptor type B1 (BMPR1B), on LSC in the tumor niche and thus sustain the survival of BMPR1B+ LSC and progenitor cells. Upon treatment, LSC and progenitor cells increase expression of BMPR1B, which progressively activate the BMP4 autocrine loop and result in TKI resistance.60 The bi-directional signal of BMP4- BMPR1B LSC alongside the Jak2-stat pathway has a crucial role in the persistence of quiescent LSC in the microenvironment. Targeting BMP and the Jak2-stat pathway has been proposed to release the LSC from quiescent state and eradicate it by TKI58 (Figure 3).\n\n\nmiRNA role in CML TKI-resistance\n\nmiRNA is a small non-coding single-stranded RNA that controls gene expression.61 Normally, miRNA-126 is expressed at a high level in hematopoietic stem cells and progenitor cells which conserve quiescence and restrain cell cycle progression. An increased level of miRNA-126 support the quiescent and engraftment of CML LSC in the bone marrow niche. However, its level found to be downregulated in CML LSC compared to normal hematopoietic stem cells. Although BCR-ABL downregulates miR-126 in CML LSC, endothelial cells of bone marrow express a high level of miR-126 that supports CML LSC and thus sustain quiescence and leukemic growth.62 Moreover, quiescent CML LSC expresses a higher level of miR-126 and more engraftment capacity than proliferating CML LSC. Interestingly, a combination of TKI and the CpG miR-126 inhibitor resulted in the elimination of LSC.61\n\nmiRNA-409-5p expression is lower in peripheral blood of CML compared to healthy children. NUP43, the targeted gene of miRNA-409-5p, has shown to be overexpressed in CML children’s patients.63 Overexpression of miRNA-409-5p downregulates the expression of NUP43 thereby inhibiting the proliferation and arresting the cell cycle. Furthermore, overexpression of miRNA-409-5p improves imatinib-induced proliferation inhibition and cell cycle arrest.63 Recently, in a study of miRNome profiling of LSC from CML-CP patients revealed a nine-fold increase of miR-196a-5p in CD34+CD38−CD26+ (BCR-ABL+) compared to CD34+CD38−CD26- (BCR-ABL-).64 Amplification of BCR-ABL and increased Jak2 signaling activate ADAR1, a double-stranded RNA protein that mediates adenosine to inosine (A-to-I) editing, thereby enhance the capacity of CML-LSC renewal by impairing the biogenesis of the Let-7miRNA family.65\n\nmiR-451, a tumor suppressor, has demonstrated downregulation in some cancer types. In CML, miR-451 expression is associated with favorable prognosis, as this miR targets ABL and BCR-ABL directly.66 In contrast, miR-21 is abundantly expressed in various solid and hematologic tumors.67 Investigating miR-21 and miR-451 levels at diagnosis of CML is of great importance as a predictor of optimal TKI response. A high level of miR-451 at diagnosis was significantly correlated to optimal response to TKI after 6 and 12 months whereas a high level of miR-21at diagnosis predicts a lower probability of reaching the optimal response to therapy.68 Inhibitors of miR-21 and PI3K together with imatinib significantly decrease AKT phosphorylation and C-MYC expression, thereby enhancing imatinib-induced apoptosis in CML-LSC.69 Pellicano et al., demonstrated that has-miR183 is highly expressed in the BCR-ABL dependent pathway that mediates inhibition early growth response 1 (EGR1) leading to upregulation of E2F1, transcription factor, and consequently, enhance the proliferation of CML-LSC.70 CML patients express a low level of miR30a compared to normal control individuals. Also, decreased level of miR30a has revealed an increase in the level of BCR-ABL that consequently enhances cell proliferation.71 Moreover, targeting miR30a enhances imatinib activity against CML mediated by Becline1and autophagy protein 5.72 Upregulation of miR29a-3p and miR660-5p target and downregulate TET2 and EPAS1, respectively, that in turn confer TKI-resistance to CML-LSC. Furthermore, downregulation of miR-494-39 in LSC induces c-MYC overexpression, which is strongly connected to TKI-resistance BCR-ABL.73\n\n\nConclusion\n\nTKIs have completely changed the treatment and improved the overall survival of CML patients. Dasatinib and bosutinib, new versions of TKIs, are considered as first-line therapy for CML patients who develop resistance to TKI (imatinib) as a result of KD mutation. CML LSC persists through TKI therapy and sustains the disease in patients. So, the determination and elimination of LSC might be of great need for cancer cure. Moreover, engraftment of CML LSCs in the bone marrow niche protect them from imatinib treatment. Therefore, inhibitors that are involved in releasing BCR-ABL+ from the bone marrow niche might increase responsiveness to TKIs. In CML, miRNA expression profile has a potential role as an effective diagnostic biomarker, monitoring disease progression and drug response. Furthermore, a biological understanding of the role of the miRNA in TKIs response and resistance still needs to be researched.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nMelo JV, Barnes DJ: Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat. Rev. Cancer. 2007; 7(6): 441–453. Publisher Full Text\n\nSloma I, et al.: Insights into the stem cells of chronic myeloid leukemia. Leukemia. 2010; 24(11): 1823–1833. 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PubMed Abstract \| Publisher Full Text\n\nBranford S, et al.: Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood. 2003; 102(1): 276–283. PubMed Abstract \| Publisher Full Text\n\nHughes T, et al.: Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood. 2006; 108(1): 28–37. PubMed Abstract \| Publisher Full Text\n\nQuintas-Cardama A, Kantarjian HM, Cortes JE: Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia. Cancer Control. 2009; 16(2): 122–131. PubMed Abstract \| Publisher Full Text\n\nWeisberg E, et al.: Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell. 2005; 7(2): 129–141. PubMed Abstract \| Publisher Full Text\n\nHughes T, et al.: Impact of baseline BCR-ABL mutations on response to nilotinib in patients with chronic myeloid leukemia in chronic phase. J. Clin. Oncol. 2009; 27(25): 4204–4210. PubMed Abstract \| Publisher Full Text\n\nCortes JE, et al.: A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias. N. Engl. J. Med. 2013; 369(19): 1783–1796. PubMed Abstract \| Publisher Full Text\n\nQuintas-Cardama A, Kantarjian H, Cortes J: Targeting ABL and SRC kinases in chronic myeloid leukemia: experience with dasatinib. Future Oncol. 2006; 2(6): 655–665. PubMed Abstract \| Publisher Full Text\n\nRedaelli S, et al.: Activity of bosutinib, dasatinib, and nilotinib against 18 imatinib-resistant BCR/ABL mutants. J. Clin. Oncol. 2009; 27(3): 469–471. PubMed Abstract \| Publisher Full Text\n\nO'Hare T, et al.: AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer Cell. 2009; 16(5): 401–412. PubMed Abstract \| Publisher Full Text\n\nCortes JE, et al.: Ponatinib efficacy and safety in Philadelphia chromosome-positive leukemia: final 5-year results of the phase 2 PACE trial. Blood. 2018; 132(4): 393–404. PubMed Abstract \| Publisher Full Text\n\nMassaro F, Molica M, Breccia M: Ponatinib: A Review of Efficacy and Safety. Curr. Cancer Drug Targets. 2018; 18(9): 847–856. PubMed Abstract \| Publisher Full Text\n\nHughes TP, et al.: Asciminib in Chronic Myeloid Leukemia after ABL Kinase Inhibitor Failure. N. Engl. J. Med. 2019; 381(24): 2315–2326. PubMed Abstract \| Publisher Full Text\n\nOzgur Yurttas N, Eskazan AE: Novel therapeutic approaches in chronic myeloid leukemia. Leuk. Res. 2020; 91: 106337. PubMed Abstract \| Publisher Full Text\n\nBurchert A, et al.: Compensatory PI3-kinase/Akt/mTor activation regulates imatinib resistance development. Leukemia. 2005; 19(10): 1774–1782. PubMed Abstract \| Publisher Full Text\n\nPacker LM, et al.: Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant chronic myeloid leukemia. Cancer Cell. 2011; 20(6): 715–727. PubMed Abstract \| Publisher Full Text\n\nXin P, et al.: Efficacy of the dual PI3K and mTOR inhibitor NVP-BEZ235 in combination with imatinib mesylate against chronic myelogenous leukemia cell lines. Drug Des. Devel. Ther. 2017; 11: 1115–1126. PubMed Abstract \| Publisher Full Text\n\nLoscocco F, et al.: BCR-ABL Independent Mechanisms of Resistance in Chronic Myeloid Leukemia. Front. Oncol. 2019; 9: 939. PubMed Abstract \| Publisher Full Text\n\nGiustacchini A, et al.: Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia. Nat. Med. 2017; 23(6): 692–702. PubMed Abstract \| Publisher Full Text\n\nEisterer W, et al.: Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease. Leukemia. 2005; 19(3): 435–441. PubMed Abstract \| Publisher Full Text\n\nHerrmann H, et al.: CD34(+)/CD38(-) stem cells in chronic myeloid leukemia express Siglec-3 (CD33) and are responsive to the CD33-targeting drug gemtuzumab/ozogamicin. Haematologica. 2012; 97(2): 219–226. PubMed Abstract \| Publisher Full Text\n\nHerrmann H, et al.: Delineation of target expression profiles in CD34+/CD38- and CD34+/CD38+ stem and progenitor cells in AML and CML. Blood Adv. 2020; 4(20): 5118–5132. PubMed Abstract \| Publisher Full Text\n\nEldesouki RE, et al.: Identification and Targeting of Thomsen-Friedenreich and IL1RAP Antigens on Chronic Myeloid Leukemia Stem Cells Using Bi-Specific Antibodies. Onco. Targets. Ther. 2021; 14: 609–621. PubMed Abstract \| Publisher Full Text\n\nBocchia M, et al.: Residual Peripheral Blood CD26(+) Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission. Front. Oncol. 2018; 8: 194. PubMed Abstract \| Publisher Full Text\n\nGodavarthy PS, et al.: The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin - SCL/TAL1 - CD44 axis. Haematologica. 2020; 105(1): 136–147. PubMed Abstract \| Publisher Full Text\n\nLaperrousaz B, et al.: Primitive CML cell expansion relies on abnormal levels of BMPs provided by the niche and on BMPRIb overexpression. Blood. 2013; 122(23): 3767–3777. PubMed Abstract \| Publisher Full Text\n\nGrockowiak E, et al.: Immature CML cells implement a BMP autocrine loop to escape TKI treatment. Blood. 2017; 130(26): 2860–2871. PubMed Abstract \| Publisher Full Text\n\nLechman ER, et al.: Attenuation of miR-126 activity expands HSC in vivo without exhaustion. Cell Stem Cell. 2012; 11(6): 799–811. PubMed Abstract \| Publisher Full Text\n\nZhang B, et al.: Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia. Nat. Med. 2018; 24(4): 450–462. PubMed Abstract \| Publisher Full Text\n\nLiu YY, et al.: MiRNA-409-5p dysregulation promotes imatinib resistance and disease progression in children with chronic myeloid leukemia. Eur. Rev. Med. Pharmacol. Sci. 2019; 23(19): 8468–8475. PubMed Abstract \| Publisher Full Text\n\nRuiz MS, et al.: miRNome profiling of LSC-enriched CD34(+)CD38(-)CD26(+) fraction in Ph(+) CML-CP samples from Argentinean patients: a potential new pharmacogenomic tool. Front. Pharmacol. 2020; 11: 612573.\n\nZipeto MA, et al.: ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis. Cell Stem Cell. 2016; 19(2): 177–191. PubMed Abstract \| Publisher Full Text\n\nYeh CH, Moles R, Nicot C: Clinical significance of microRNAs in chronic and acute human leukemia. Mol. Cancer. 2016; 15(1): 37. PubMed Abstract \| Publisher Full Text\n\nFeng YH, Tsao CJ: Emerging role of microRNA-21 in cancer. Biomed Rep. 2016; 5(4): 395–402. PubMed Abstract \| Publisher Full Text\n\nAlves R, et al.: MicroRNA signature refine response prediction in CML. Sci. Rep. 2019; 9(1): 9666. PubMed Abstract \| Publisher Full Text\n\nWang WZ, et al.: Silencing of miR-21 sensitizes CML CD34+ stem/progenitor cells to imatinib-induced apoptosis by blocking PI3K/AKT pathway. Leuk. Res. 2015; 39(10): 1117–1124. PubMed Abstract \| Publisher Full Text\n\nPellicano F, et al.: hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival. Blood. 2018; 131(14): 1532–1544. PubMed Abstract \| Publisher Full Text\n\nLiu Y, et al.: Decreased microRNA-30a levels are associated with enhanced ABL1 and BCR-ABL1 expression in chronic myeloid leukemia. Leuk. Res. 2013; 37(3): 349–356. PubMed Abstract \| Publisher Full Text\n\nYu Y, et al.: Targeting microRNA-30a-mediated autophagy enhances imatinib activity against human chronic myeloid leukemia cells. Leukemia. 2012; 26(8): 1752–1760. PubMed Abstract \| Publisher Full Text\n\nSalati S, et al.: Deregulated expression of miR-29a-3p, miR-494-3p and miR-660-5p affects sensitivity to tyrosine kinase inhibitors in CML leukemic stem cells. Oncotarget. 2017; 8(30): 49451–49469. PubMed Abstract \| Publisher Full Text" }	[ { "id": "118639", "date": "24 Jan 2022", "name": "Ala-Eddin Al Moustafa", "expertise": [ "Reviewer Expertise Yes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 author reviewed the role of the BCR-ABL gene and its inhibitor (TKI therapy) in the response and resistance of Chronic myeloid leukemia (CML). This is an interesting review paper that can be accepted for publication after some revisions as follows:\nThe author should start the introduction with a paragraph about leukemia in general and its different categories, including CML.\n\nThe author should remove Figure 1 from the paper which is not necessary.\n\nThe author should replace Figure 2 with a table.\n\nIn the conclusion section, “miRNA expression profile has a potential role as an effective diagnostic biomarker” the word “expression” should be removed and replaced by “miRNA profile, particularly circulating …”\n\nThe paper needs some revision for English.\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": "7785", "date": "18 Feb 2022", "name": "Mohammad Al Hamad", "role": "Author Response", "response": "A paragraph about leukemia in general and its different categories is added. Figure 1 is removed from the paper. Figure 2 is replaced with a table. “expression” is removed and replaced by “miRNA” profile. Revision for English is done." } ] } ]	1	https://f1000research.com/articles/10-1288
https://f1000research.com/articles/11-164/v1	09 Feb 22	{ "type": "Software Tool Article", "title": "NIFtHool: an informatics program for identification of NifH proteins using deep neural networks", "authors": [ "Jefferson Daniel Suquilanda-Pesántez", "Evelyn Dayana Aguiar Salazar", "Diego Almeida-Galárraga", "Graciela Salum", "Fernando Villalba-Meneses", "Marco Esteban Gudiño Gomezjurado", "Jefferson Daniel Suquilanda-Pesántez", "Evelyn Dayana Aguiar Salazar", "Diego Almeida-Galárraga", "Graciela Salum" ], "abstract": "Atmospheric nitrogen fixation carried out by microorganisms has environmental and industrial importance, related to the increase of soil fertility and productivity. The present work proposes the development of a new high precision system that allows the recognition of amino acid sequences of the nitrogenase enzyme (NifH) as a promising way to improve the identification of diazotrophic bacteria. For this purpose, a database obtained from UniProt built a processed dataset formed by a set of 4911 and 4782 amino acid sequences of the NifH and non-NifH proteins respectively. Subsequently, the feature extraction was developed using two methodologies: (i) k-mers counting and (ii) embedding layers to obtain numerical vectors of the amino acid chains. Afterward, for the embedding layer, the data was crossed by an external trainable convolutional layer, which received a uniform matrix and applied convolution using filters to obtain the feature maps of the model. Finally, a deep neural network was used as the primary model to classify the amino acid sequences as NifH protein or not. Performance evaluation experiments were carried out, and the results revealed an accuracy of 96.4%, a sensitivity of 95.2%, and a specificity of 96.7%. Therefore, an amino acid sequence-based feature extraction method that uses a neural network to detect N-fixing organisms is proposed and implemented. NIFtHool is available from: https://nifthool.anvil.app/", "keywords": [ "Deep Neural Network", "Embedding Layers", "NifH protein", "Software", "k-mers" ], "content": "Introduction\n\nNitrogen is an essential nutrient for plants. Nitrogen fertilizers have the highest worldwide demand during agricultural practices.1,2 Among the vast diversity of microorganisms, different bacterial taxa have developed the capacity to use atmospheric nitrogen as a substrate to produce ammonia (NH4) through the biological nitrogen fixation (BNF) process.3 BNF is the most critical pathway of incorporating N-NH4 inside the biosphere.4 Estimating that only 10% of the total nitrogen incomes proceed from atmospheric precipitation, the rest is through this biological process.5\n\nThe activity of the nitrogenase enzyme carry out the BNF. This enzyme is a molecular complex constituted by two subunits: (i) the dinitrogenase reductase (NifH), which is an iron protein that participates in the electrons transport from ferredoxins, to the (ii) dinitrogenase or molybdenum-iron-protein.3,6 Molybdenum-iron-protein (Mo-Fe) is the catalytic site, which catalyses the N2 reduction using 16 ATP molecules as an energy source.7 Both units are encoded in the nifHDK operon (genes encoding for the Fe/Mo-Fe nitrogenase protein complex) located on the bacterial chromosome or plasmids, depending on the bacterial species8 (Figure 1). The principal role of NifH protein is donating electrons to molybdenum-iron proteins (NifD/NifK), favouring N2 reduction. Efficient and quick identification of NifH proteins is of relevant interest because Nitrogen engineering focuses on the development of new products during farming activities.9\n\nThe nifHDK operon (genes encoding for the Fe/Mo-Fe nitrogenase protein complex) codifies for the subunits of the nitrogenase enzyme, which catalyzes the reduction of the N2 to NH4 in an ATP- dependent manner through the electron flux from the dinitrogenase reductase to the molybdenum-iron (Mo-Fe) protein subunit. Modified from Ref. 10.\n\nPrevious studies have focused on developing new informatics tools to identify nifH genes from different bacterial genera. Frank et al. (2016) retrieved the sequences of the nifH genes in the genome of nitrogen-fixing microorganisms and achieved the classification of the nifH sequence at different clusters.11 Likewise, Shinde et al. (2019) developed his nifH genes classification model based on image processing and convolutional neural network.12 On the other hand, Frank (2014) designed a tool with sufficient information to carry out phylogenetic cluster membership predictions from 32954 NifH protein sequences.13 These three studies obtained good results. However, their investigations did not produce a computer tool.\n\nMeher et al. (2018) developed nifPred, a machine learning (ML) software, to perform a sequence classification into NifH or non-NifH proteins. This informatics tool converts multi-categorical sort of gene sequences into one of the six types of the Nif proteins encoded by the nif operon using a high computational performance.14\n\nConstant supervision is necessary to guide the program in all phases of the system, which causes the increment of computational cost.15 Nowadays, some algorithms are registered in literature to make predictions of NifH proteins based on gene sequences. However, there is still no tool to distinguish Nif proteins from amino acids sequences.\n\nBased on this background, the objective of this work was to develop an informatics tool that uses deep neural networks with the lowest computer cost to play an essential role in the improvement of the BNF process research through the identification of the NifH proteins among different bacterial genera in a reliable way.\n\n\nMethods\n\nThe proposed model was developed into five main stages: (i) acquisition of raw data from the UniProt protein databank 16 (Universal Protein Resource, RRID:SCR_002380); (ii) feature extraction stage, which allows defining numerical vectors as representation of amino acids (aa) sequences; (iii) development of a prediction model or classifier using a deep neural network (DNN); (iv) K-fold Cross-validation to evaluate the model and (v) the identification of the predicted label of the sequence (Figure 2).\n\ni) Data acquisition, ii) Feature extraction, iii) Modeling of deep learning, iv) K-fold Cross-validation and v) prediction and providing information. Dinitrogenase reductase = NifH.\n\nExperimental processes were carried out using an Intel Core i5-8100 processor machine, feature extraction process and neural network code were written in Python v. 3.8 language (RRID:SCR_008394), and the classifier was implemented on Anvil Works.17\n\nThe binary classification of the protein sequences was arranged under two types of labels: 1 (NifH) and 0 (non-NifH). The raw dataset was constructed by NifH and non_NifH protein sequences extracted from database UniProt18 in the FASTA format up to March 10th 2021.18 NifH and non-NifH sequences were retrieved by searching for ‘NifH, nifH proteins’ and ‘non-NifH, non-nifH proteins’, respectively, considering the following parameters: organism identification, gen name, proteins names, and length. Uniprot provided 52942 NifH proteins and 5763 non-NifH at the end of the search.\n\nCD-HIT software (RRID:SCR_007105) analysed the raw dataset19 to remove redundant sequences with 90% similarity to avoid undesirable biases. Next, positive, and negative sequences, 4939 and 4953, respectively, were obtained after the cleaning process. Sequences were filtered for lengths greater than 50 aa and shorter than 1173 aa, the maximum length of NifH sequence. These values were set up for two reasons: (i) the upper limit because non-nifH sequences are greater than 1173 aa and (ii) shorter sequences than a defined upper limit are padded with zeros until the limit is reached that guarantees the conversion of aa sequences into numeric vectors during the embedding layers (feature extraction stage). For instance, for a maximum value defined as 2000, a 125 aa sequence would have to be completed with 1875 zeros; for a sequence of 1750 aa, it would only be necessary to complete it with 250 zeros. The redundancy of a large amount of zeros can be a factor leading to undesirable bias.20 Thus, 4911 NifH and 4782 non-NifH protein sequences were obtained after filtration of limits to build the final dataset of 9693 sequences (Figure 3).\n\nThe curves show the correlation between the number of sequences and the number of amino acids of dinitrogenase reductase (NifH) and non-NifH proteins.\n\nThis step results in 328-feature numeric vectors representing each of the sequences. The values were obtained through two different processes, k-mers, which are related to the presence of specific groups, and embedding vectors, related to the location of the amino acids in the sequence.\n\nk-mers\n\nThis analysis allows the sequence representation based on the presence of specific groups called k-mers, where k is the length of that group.21 For example, 5-mers represents a specific group of 5 amino acids. This stage begins with the generation of a list of the most common k-mers within the NifH dataset. Considering 5-mers, for each sequence of the dataset of 4939 NifH proteins, all the different 5-mers are obtained and their quantities are counted. Then, all the 5-mers in the entire dataset are identified and the k-mers with the highest frequency are defined, which form the general list (GL) of 5-mers where the order of each value is relevant. To generate a numerical vector for a certain sequence, GL is compared with that sequence and according to the presence of the GL 5-mers in the sequence, ‘1’ is recorded for presence and ‘0’ for absence according to the order of the GL22 (Figure 4).\n\nThis process comprised some sequential steps: 1) Input of the amino acid (aa) sequences, 2) extraction, and 3) development of the convolutional layer. Where M is the length of the input sequence and L is the length of the embedding vector.\n\nFor instance, in the sequence ‘GAHYTGGTPLNFH’ nine 5-mers can be identified, some examples of the 5-mers are GAHYT, AHYTG, HYTGG, PLNFH. If the GL of 5-mers were made up of 5 k-mers, for example: AGHLP, PLNFH, HJKLP, YTGGT and TGHHT the resulting vector for the example sequence would be [0, 1, 0, 1, 0]. The length of the vector is five because the length of the GL is five. According to the order, each GL 5-mer is searched in the sequence and if the 5-mer is present, 1 is recorded. In the example, the vector has ‘1’ at position 2 because PLNFH is present in the sequence and is the second most common of the GL.\n\nEmbedding features\n\nThis section was performed as described previously by Shadab et al. (2020) and comprised the carrying on: (i) input sequences, (ii) embedding vectors, and (iii) convolutional layers23 (Figure 4).\n\nInput sequences\n\nProtein sequences have different lengths depending on the number of aa. Deep learning requires the same length for all sequences.24,25 The maximum size determined from the data had a value of 1173. Each number sequence with a length less than this value goes through a filling process, and the sequence was filled with the number “0” (token) until the maximum size is achieved (Figure 4). These zero vectors do not affect the output of the subsequent layers, and M was defined as the length of the input sequence.23,24 In addition, the aa count per chain was performed, and this set presented a minimum of 50, a maximum of 1173, and an average of 278.43 (Figure 3).\n\nEmbedding vectors\n\nPadded data were passed throughout an embedding layer to get a dense vector. This layer is used to transform discrete inputs into points in a vector space, called embedding vectors (L is the length of this vector).23 Each aa of the protein sequences, both in the training and test sets, had a specific integer number, and the result of the encoding phase is the integer number vectors. For instance, in the sequence ‘ACLKIGAL’, a possible encoding would be ‘1-4-6-8-13-15-1-6’ (Figure 4). This algorithm randomly assigns the digits but maintains the same number for a specific aa. The order in which the numbers are assigned could be a relevant aspect regarding the model's performance. However, it has been proved that order does not influence the result.23,25 The final output of the embedding layers is a uniform matrix of size L×M.\n\nConvolutional layer\n\nA trainable convolutional layer was added, and its input was a uniform matrix (embedding vectors). This layer applied convolution using 128 trainable filters, each with a size of L×31. The result was 128 feature maps, each of the same size. To reduce overfitting and capture noise, we used a max-pooling (window size of 3×3) to subsample these feature maps.23 Finally, this feature map was flattened into a 1×1 dimensional matrix, where each matrix represents the features of the input sequence (Figure 4). These features were used to train the classifiers.\n\nThe advantage of this method relies on the results of the classification model that can be back-propagated to the convolution and embedding layers.23,25,26 These layers were trained to extract better features.\n\nThis stage allows the determination of the the most efficient dataset to perform the training and evaluation of the neural network. For this purpose, different methodologies are used to create the datasets. Two techniques for feature extraction were considered, the methodologies will focus on the combination of these techniques and the number of features that are extracted. Due to the embedding vectors (EV) has 128 fixed features, the variation of dataset methodologies depends on the length of k-mers and number of k-mers in the GL. Some datasets were EV, 3-mers (100 features) + EV, 7-mers (300 features) + EV, 3-mers (100 f) + 5-mers (100 f) + EV, and so on. Some datasets were created by combining k-mers and EV, but others were created by combining two different k-mers and EV. Table 2 shows the different methodologies analysed. After a series of analysis with the dataset methodologies to determine the most optimal number of features to compose the numeric vectors the feature extraction stage results in vectors of 328 numeric values corresponding to 100 5-mer features, 100 features of 7-mers, and 128 values of embedding vectors (Figure 4).\n\nFor the prediction of the identity of the aa sequence, a Deep Neural Network (DNN) was designed. Its input corresponds to the representation of the sequences (array of 328 numbers) and the output is the class of each sequence: 1 (NifH) and 0 (non-NifH). This DNN was written in Python v.3.8 using the following libraries: i) Pandas (RRID:SCR_018214),27 ii) Keras,28 iii) Scikit-learn (RRID:SCR_002577),29 iv) NumPy (RRID:SCR_008633),30 and v) Matplolib.31 First, the hyperparameters of our model were related to the learning algorithm level: training of 40 epochs at 6 seconds, using a batch size of 40 and a learning rate of 6x10-5. Second, hyperparameters related to structure and topology were the layers. The deep learning model consisted of 12 layers, excluding the input and output layers (Figure 5).\n\nIt was composed of four blocks, and the number of neurons for each block was 128, 64, 32, and 2, from the first to last one, respectively.\n\nThe model established the number of neurons in each defined block of layers, being 128 neurons for the first two layers, 64 neurons for the following four layers, followed by 32 neurons for the next four layers, and finally, two neurons at the last two. The number of neurons was placed according to the input parameters and the architecture of the DNN.32 There were four layers in the neural network corresponding to the dense, activation, dropout, and batch normalization layers.26,33 Four layers were used: (i) four dense layers, (ii) three activation layers, (iii) three dropout Layer, and (iv) two batch normalisation layers. The detailed configuration and order of layers of the proposed DNN model are shown in Table 1.\n\nTo evaluate the classification accuracy of the several dataset methodologies considered to train the neural network (Table 2), the k-fold cross-validation technique was performed. k-fold cross-validation divides datasets into k-subsets and requires that each subset is used to validate data exactly once.34 In this validation process, k typically is 10, even though to reduce the computational time, this study used k = 4, giving 4-fold cross-validation (Figure 6).\n\nData were divided into four folders, three were used to train the classifier, and one was allowed to test during each of four iterations.\n\nDatasets were partitioned into four equal parts; one group was used to test the methodologies (test dataset), and the remaining groups were used to train the software (Train Dataset). Four iterations were needed for each part to validate the methodologies throughout an accuracy and confusion matrix obtained for each iteration to discard the model. Then, each dataset methodology is processed by 4 iterations, where induvial iteration generates a classifier. Thus, when the classifier with the best performance is found, this is saved and used to predict NifH proteins.\n\nNIFtHool requires access to internet and to have any device capable of being a Web server. The device must have an operating system that can run as a Web server, capable of delivering HTML5 content. It must also have an Intel® Celeron® 847 Processor, 1.10 GHz, and a minimum Ram of 512 MB. Finally, this tool does not require the device to have a certain amount of storage or hard disk space as it works online.\n\n\nResults\n\nPerformance of four-fold cross-validation and metrics such as accuracy, loss, F1-score, sensitivity or recall, and precision were obtained for each dataset methodology to evaluate the classification as summarized in Table 2. Due to each dataset methodology works with four iteration producing four classifiers, only the one with the best performance is mentioned below.\n\nThe use of the methodology that consists of EV had a high performance, since both its precision, recall and F1-score were greater than 90%. However, its loss still represents a significant value. For this reason, EV was merged with k-mers to increase the classification values. A large number of methodological datasets were tested, and all of them were higher than the EV. The results define that datasets with longer k-mers, such as 20-mers, have slightly lower performance than datasets with shorter k-mers, such as 7-mers. Because the computational cost is higher as the k-mers are longer, and because their performance decreases slightly, shorter k-mers were selected to improve the classification performance.\n\nAnother factor considered when comparing the different methodologies was the number of features extracted. For the 3-mers + EV and 7-mers + EV datasets, the extraction of 100, 200 and 300 features were tested, but the results were similar between the groups. In the group of 3-mers + EV, the accuracy values were around 95% for the three cases, as well as recall and F1-score, which were the same (96%) for all. For the group of 7-mers + EV, despite being k-mers of greater length than the 3-mers, they obtained similar results to 3-mers + EV, where their greatest difference was the loss, which represents an advantage for the 7-mers with values between 13 and 17%, compared to the higher 17 to 21% of the 3-mers. In this sense, since a greater number of features implies a higher computational cost, and the values are similar, the extraction of 100 features for each k-mer was chosen. The analysis of 5-mers + EV was also recorded, which was similar to 3-mers + EV, however this analysis showed better performance in the loss, since 5-mers reached 13% while the best result of 3-mers was 17%.\n\nMethodologies that combine two k-mers + EV were experimented with, and due to the high performance of each k-mer together with EV, the performance of the combination of these k-mers with EV was analysed. Finally, five dataset methodologies were performed, and 5-mers (100 features) + 7-mers (100 features) + EV dataset had the best performance, which obtained precision, recall and F1-scores of 96%, an accuracy of 96.37%, and a loss of only 14.79%. Due to its performance, this methodology was selected to be used as the model classifier and to be implemented into an informatics tool.\n\nThe validation error of model 2 was 0.2625. This value was obtained from the learning rate, ranging from 0 to 40 in which the red epochs were trained. Measurements showed suitable training convergences as shown in Figure 7. Accuracy evaluation of the training started with low values that increased in the epochs (Figure 7b). On the other hand, while training began with a high loss, this value decreases as they were trained (Figure 7a). Both graphs show similar behaviour during the training and validation as a reliable model learning with constant values obtained at the end of the assessment, which indicates that the maximum training point was reached.\n\nWhere x-axis represent the number of epochs and the y-axis represents the values of accuracy and loss as a function of unity (1= 100%). a) Loss evaluation. b) Accuracy evaluation.\n\nThe efficiency of the neural network was assessed using a confusion matrix. Primary diagonal data was represented, which indicates the number of hits in the model (Figure 8). 1195 sequences were correctly classified as no-NifH, and 1128 as true NifH proteins. The value below the primary diagonal shows the false negatives or type II errors (the NifH is not detected), corresponding to 61 cases.\n\na) Panel a shows the confusion matrix for the number of evaluated sequences, and panel b corresponds to the number of evaluated sequences normalized to one. TP: true positive =1188, TN: true negative = 1132, FP: false positive = 25, and FN: false negative = 48.\n\nIn contrast, the value above the primary diagonal reflected the classifier errors: false positives or error type I (the NifH is detected but not present) was equal to 39 cases. The confusion matrix results evaluated the relevance through 3 metrics: accuracy rate (96.4%), specificity (96.7%), and sensitivity (95.2%).\n\n\nDiscussion\n\nDeep learning was selected because it worked with protein sequence and conversion to arrays allowing better results than other methods, as previously reported (Table 3). Our model had a high performance, as demonstrated high sensitivity (95.2%), high accuracy (96.4%), and specificity (96.7%), which is comparable to current deep learning techniques generating software23,25 that considered a vector of 128 features-values for each sequence protein to train the model.\n\n§ ANN: Artificial neural network.\n\n¶ CNN: Convolutional neural network.\n\n¥ SVM: Support vector machine.\n\n£ MBD-LSTM: Multilayer bi-directional long short term memory.\n\nÞ DeepDBP-ANN: Deep neural networks for identification of DNA binding proteins.\n\n† CART: Classification and regression trees statistical models.\n\n* NN: Neural networks.\n\n** ML: Machine leaning.\n\n€ NifH: Nitrogenase Iron Protein.\n\nγ NifD: Nitrogenase molybdenum-iron protein alpha chain.\n\nɑ NifK: Nitrogenase molybdenum-iron protein beta chain.\n\n∞ NifE: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE.\n\n∂ NifN: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifN.\n\nℇ NifB: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifB, N/D: No data.\n\nOur software showed a high performance as compared with previous apps. For instance, Shinde's model12 is based on the analysis of gene sequences of NifH proteins, operated with a 32×32 matrix for each sequence as our model. Nonetheless, our software is an improvement on previous informatics tools as it uses other feature extraction methods, as described above. nifPred, a multi NifH proteins classifier, that uses 13,500 values per sequence, involves four manual methods to obtain the components and data to be trained, having a high specificity.14\n\nNIFtHool was compared with two models that work with two different embedding vectors to identification of mitochondrial proteins of Plasmodium falciparum25 and DNA-binding proteins.23 The three models showed positive results in sensitivity, specificity, and accuracy, so the selection of the embedding vector method was adequate. Additionally, a comparison was made with studies using machine learning techniques for the classification of NifH protein sequences. These studies used classification and regression trees statistical models and decision trees.11,13\n\nIt is stated that our model obtained high accuracy results like these models but with reduced computational power. Thus, NIFtHool shows clear improvements compared to the studies in the literature as shown in Table 3.\n\n\nConclusion\n\nA binary classification model of NifH protein sequences using artificial neural networks has been developed in the present work and hosted by Anvil. We tried the conventional approach of extracting features with specified algorithms through the novel feature extraction approach using deep learning techniques. Numerical features were obtained from two aspects: k-mers method considering the unique k-mer and an embedding layer related to aa position in the sequence. This methodology that was studied offers a performance similar to the best performances in the literature. Our tool offers better computational performance due to the classification process being based on the use of only NifH protein domain, resulting in less data processing for the software.\n\nEven though the entire nitrogenase complex is relevant for transforming atmospheric nitrogen into ammonia, only NifH protein has been considered because this subunit is paramount during the reduction from N2 to NH4. NIFtHool not only represents a significant improvement compared to other computational methods, but it is also a tool for the identification of NifH Protein. Thus, researchers can easily use NIFtHool to identify NifH proteins as a reliable tool during the protein and nitrogen fixing bacteria analysis.\n\n\nData availability\n\nZenodo: NIFTHool: Repository. https://doi.org/10.5281/zenodo.5913032.18\n\nThis project contains the following underlying data:\n\n- List_kmers.csv (List of 5-mers and 7-mers obtained from dataset after it filtered sequences shorter than 50 aa and longer than 1173 aa)\n\n- RAW_data_NifH.fasta (52942 NifH proteins retrieved from Uniprot)\n\n- data_NifH.csv (4939 NifH sequences retrieved after CD-hit filtration)\n\n- data_nonNifH.txt (4953 non-NifH sequences)\n\nZenodo: NIFTHool: Repository. https://doi.org/10.5281/zenodo.5913032.18\n\nThis project contains the following underlying data:\n\n- data_NifH_plus_nonNifH.txt (Combination of sequences from data_NifH.csv and data_nonNifH.txt).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nNIFtHool available from: https://nifthool.anvil.app/\n\nSource code available from: https://github.com/JefferDSP/NIFTHool/tree/v1.0\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.5913032.18\n\nLicense: CC0-1.0.", "appendix": "References\n\nCao P, Lu C, Yu Z: Historical nitrogen fertilizer use in agricultural ecosystems of the contiguous United States during 1850-2015: Application rate, timing, and fertilizer types. Earth Syst. Sci. Data. 2018; 10(2): 969–984. Publisher Full Text\n\nFAO: World fertilizer trends and outlook to 2020. Food and Agriculture Organization of United Nations; 2017. Reference Source\n\nMahmud K, Makaju S, Ibrahim R, et al.: Current progress in nitrogen fixing plants and microbiome research. Plants. 2020; 9(1): 1–17. Publisher Full Text\n\nBhattacharjee RB, Singh A, Mukhopadhyay SN: Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: Prospects and challenges. Appl. Microbiol. Biotechnol. 2008; 80(2): 199–209. PubMed Abstract \| Publisher Full Text\n\nDavies-Barnard T, Friedlingstein P: The Global Distribution of Biological Nitrogen Fixation in Terrestrial Natural Ecosystems. Glob. Biogeochem. Cycles 2020; 34: 1–14. Publisher Full Text\n\nSun W, Shahrajabian MH, Cheng Q: Nitrogen Fixation and Diazotrophs – A Review. Rom. Biotechnol. Lett. 2021; 26(4): 2834–2845. Publisher Full Text\n\nBellenger JP, Darnajoux R, Zhang X, et al.: Biological nitrogen fixation by alternative nitrogenases in terrestrial ecosystems: a review. Biogeochemistry. 2020; 149(1): 53–73. Publisher Full Text\n\nPoole P, Ramachandran V, Terpolilli J: Rhizobia: From saprophytes to endosymbionts. Nat. Rev. Microbiol. 2018; 16(5): 291–303. PubMed Abstract \| Publisher Full Text\n\nJiang X, Payá-Tormo L, Coroian D, et al.: Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants. Commun. Biol. 2021 Jan; 4(1): 1–11. Publisher Full Text\n\nRascio N, La Rocca N: Biological Nitrogen Fixation. In: Fath B, editor. Encyclopedia of Ecology. Second edition.Amsterdam: Elsevier; 2019; Volume 2. : p. 264–279. Publisher Full Text\n\nFrank IE, Turk-Kubo KA, Zehr JP: Rapid annotation of nifH gene sequences using classification and regression trees facilitates environmental functional gene analysis. Environ. Microbiol. Rep. 2016 Oct; 8(5): 905–916. PubMed Abstract \| Publisher Full Text\n\nShinde I: Nitrogenase Iron Protein Detection using Neural Network. Master’s Projects. [San Jose, CA, USA]: San José State University; 2019.\n\nFrank I: Rapid Classification of NifH Protein Sequences using Classification and Regression Trees. University of California Santa Cruz; 2014.\n\nMeher PK, Sahu TK, Mohanty J, et al.: nifPred: Proteome-Wide Identification and Categorization of Nitrogen-Fixation Proteins of Diaztrophs Based on Composition-Transition-Distribution Features Using Support Vector Machine. Front. Microbiol. 2018 May; 9: 1–16. Publisher Full Text\n\nZhang X-D: Machine Learning. A Matrix Algebr Approach to Artif Intell.2020; 223–440.\n\nThe Uniprot Consortium: UniProt: The universal protein knowledgebase in 2021. Nucleic Acids Res. 2021; 49(D1): D480–D489. PubMed Abstract \| Publisher Full Text\n\nAnvil TM. Anvil Full Stack web apps with nothing but Phyton[Internet]. 2020 [cited 2022 Jan 31]. https://anvil.works/\n\nJefferson S-P: JefferDSP/NIFTHool: NIFTHool repository. NIFTHool Repository 2021 [cited 2022 Jan 31]. Publisher Full Text\n\nFu L, Niu B, Zhu Z, et al.: CD-HIT: Accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012; 28(23): 3150–3152. PubMed Abstract \| Publisher Full Text\n\nBursteinas B, Britto R, Bely B, et al.: Minimizing proteome redundancy in the UniProt Knowledgebase. Database J. Biol. Databases Curation. 2016 Jan; 2016: 1–9. Publisher Full Text\n\nManekar SC, Sathe SR: Estimating the k-mer Coverage Frequencies in Genomic Datasets: A Comparative Assessment of the State-of-the-art. Curr. Genomics 2019 Oct; 20(1): 2–15. PubMed Abstract \| Publisher Full Text\n\nBreitwieser FP, Baker DN, Salzberg SL: KrakenUniq: confident and fast metagenomics classification using unique k-mer counts. Genome Biol. 2018 Nov; 19(1): 198. PubMed Abstract \| Publisher Full Text\n\nShadab S, Alam Khan MT, Neezi NA, et al.: DeepDBP: Deep neural networks for identification of DNA-binding proteins. Informatics Med. Unlocked. 2020; 19: 100317–100318. Publisher Full Text\n\nElAbd H, Bromberg Y, Hoarfrost A, et al.: Amino acid encoding for deep learning applications. BMC Bioinformatics. 2020 Jun; 21(1): 1–14. Publisher Full Text\n\nKhan SU, Baik R: MPPIF-Net: Identification of Plasmodium Falciparum Parasite Mitochondrial Proteins Using Deep Features with Multilayer Bi-directional LSTM. Processes. 2020 Jun; 8(6): 1–16. Publisher Full Text\n\nAbadi M, Agarwal A, Barham P, et al.: TensorFlow: Large-Scale Machine Learning on Heterogeneous Distributed Systems. OSDI’16: Proceedings of the 12th USENIX conference on Operating Systems Design and Implementation. Savannah: USENIX Association; 2016; p. 265–283. Reference Source\n\nMcKinney W: Data Structures for Statistical Computing in Python. Proc 9th Python Sci Conf. 2010; 1: 56–61.\n\nKeras CF. 2015. Reference Source\n\nPedregosa F, Varoquaux G, Gramfort A, et al.: Scikit-learn: Machine Learning in Python. J. Mach. Learn. Res. 2011; 12: 2825–2830.\n\nHarris CR, Millman KJ, van der Walt SJ , et al.: Array programming with NumPy. Nature. 2020; 585(7825): 357–62. PubMed Abstract \| Publisher Full Text\n\nHunter JD: Matplotlib: A 2D graphics environment. Comput. Sci. Eng. 2007; 9(3): 90–95. Publisher Full Text\n\nCichy RM, Kaiser D: Deep Neural Networks as Scientific Models. Trends Cogn. Sci. 2019 Apr; 23(4): 305–317. Publisher Full Text\n\nSrivastava N, Hinton G, Krizhevsky A, et al.: Dropout: A Simple Way to Prevent Neural Networks from Overfitting. J. Mach. Learn. Res. 2014; 15: 1929–1958.\n\nAli M, Son D-H, Kang S-H, et al.: An Accurate CT Saturation Classification Using a Deep Learning Approach Based on Unsupervised Feature Extraction and Supervised Fine-Tuning Strategy. Energies. 2017 Nov; 10(11): 1830. Publisher Full Text" }	[ { "id": "123112", "date": "22 Mar 2022", "name": "Juan Onofre Orozco-López", "expertise": [ "Reviewer Expertise Artificial neural networks are used mainly to develop or improve the performance of the artificial pancreas in the treatment of diabetes Mellitus type 1. Also", "I used to develop automatic control algorithms", "some of them using artificial neural networks" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 rationale for developing the new software tool clearly explained? The authors explained the reasons to develop this new tool, and they also is showed a summary of existing ones and their disadvantages with the current proposal. But it would be better to justify the use of this new tool from the point of view of getting extra information together at the classification capability.\nOn the other hand, there are enough technical details on the requirements, the authors also widely explained the internal structure of Deep Neural Network (DNN), but didn't include information on how such a DNN is performed, additionally to the number of neurons per layer and the layers. It would be great if the authors can share or show a smaller or simpler version of such DNN, in order to replicate their experiments locally, because I found this next error while using the tool on the platform: \"NoServerFunctionError: No server function matching \"function_pred_seq\" has been registered\" Maybe this error is caused by the lack of information or instructions to use the tool.\nMinor comments:\nOn page 4 it appears \"NifH, nifH proteins\" was written, where I understand that capital letters refer to specific chemical elements, and the lower case \"n\" in nifH could be a mistake.\n\nFigure 6 is detailed, how is the dataset divided into train or test datasets? It would be interesting to use an extra set or save an extra 10% of the training datasets to try the tool as a real test using real data. As the whole data set was used for training purposes at different iterations, the neural network has not been tested with any non-training data at any iteration. I propose to save some data sets and use them with the goal to test the performance of the neural network with unknown data by the algorithm.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "123174", "date": "25 Mar 2022", "name": "Lorena Isabel Barona López", "expertise": [ "Reviewer Expertise Security", "Machine learning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have built a system that allows researchers the identification of the NifH proteins using deep learning. The paper is well written and explained clearly. Some unique suggestions would be to provide an explanation about how the number of samples was calculated, why was k-fold validation used and how were the hyperparameters determined? I think that these points will enhance the paper.\nBest regards\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": [] } ]	1	https://f1000research.com/articles/11-164
https://f1000research.com/articles/11-162/v1	09 Feb 22	{ "type": "Research Article", "title": "Anxiety level among newly hired nurse in a specialized oncology hospital: An observational study", "authors": [ "Ahmad Alhroub", "Hebah Albakri", "Hedaia Al-Awaysheh", "Aladeen Alloubani", "Ahmad Alhroub", "Hebah Albakri", "Hedaia Al-Awaysheh" ], "abstract": "Background: Anxiety is common among oncology nurses due to the complexity of oncology patients' needs and demands. The current study aimed to assess the anxiety level among newly hired nurses in a specialized oncology hospital throughout their initial period of employment, deploying a General Nursing Orientation (GNO) and the Clinical Resource Nurse (CRN) role. Methods: A longitudinal one-group repeated measure design was used. Participants' demographics sheet and Sate-Trait Anxiety Inventory for Adults questionnaire were used. A total of 181 newly hired nurses participated in the study. Results: The anxiety level among newly hired oncology nurses was (mean=38.65, SD=9.58) at the beginning of GNO, and the level of anxiety was highest after 90 days of employment (mean=45.71, SD=7.20). The level of anxiety among newly hired oncology nurses increased gradually from day one of the GNO, the last day of GNO, and finally, after 90 days of employment. Conclusions: Nurses working in oncology workplaces face anxiety. It is important to seek nursing administrators' attention to apply proper strategies to decrease the anxiety level among newly hired nurses to help them smoothly fit into a new team to ensure safe patient care.", "keywords": [ "Anxiety Level", "Registered Nurses", "General Nursing Orientation", "Specialized Oncology Hospital" ], "content": "Abbreviations\n\nANOVA: Analysis of variance\n\nCRN: Clinical Resource Nurse\n\nGNO: General Nursing Orientation\n\nHAS: Hamilton Anxiety Scale\n\nHCAC: Health Care Accreditation Council\n\nIDRAAC: Institution for Development Research Advocacy and Applied Care\n\nJCI: Joint Commission International\n\nSPSS: Statistical Package for the Social Sciences\n\n\nIntroduction\n\nAnxiety is defined as “something felt, an emotional state that involved feelings of apprehension, tension, nervousness, and worry followed by physiological arousal” (Freud, 1977). Freud stated that anxiety is an adaptive behavior that helps individuals cope with stressful situations. Intense anxiety is commonly associated with many psychiatric diseases. Cattell highlighted the types of anxiety, including emotional state and personality trait anxiety (Cattell, 1966).\n\nA systematic review identified 87 studies published between 1980 to 2009 across 44 countries found that the global prevalence of anxiety rate after adjusted methodological differences was 7.3% (Baxter et al., 2013). Another systematic review conducted by the Institution for Development Research Advocacy and Applied Care (IDRAAC) reviewed epidemiological anxiety disorders studies among Arab countries; the results revealed that anxiety disorder is common among the participants. Therefore, they concluded the importance of having studies about anxiety disorder and its impact on individuals and communities (Tanios et al., 2009). Several studies assessed anxiety rates amongst some Arab populations; the results showed that the anxiety rate in Saudi Arabia was 16%, 16.7% in Lebanon, and 28.2% in Jordan (Karam et al., 2008; Tanios et al., 2009).\n\nAnxiety is common among healthcare professionals, including nurses (Chen et al., 2016; Cheung et al., 2016; Creedy et al., 2017; Huang et al., 2018; Wang & Zhang, 2017). Nurses represent a large portion of healthcare professionals; nursing is a stressful profession by its nature (Roberts & Grubb, 2014). The workload, caring for sick patients, and work-related conflict with other healthcare providers are a few factors that cause nursing anxiety (Jafari et al., 2018). Moreover, the demand related to the tasks nurses perform at different shifts, the types of patients, and the lack of resources cause nursing job-related anxiety (Khodadadi et al., 2016; Shen et al., 2016).\n\nAnxiety is also more evident among oncology nurses; they may encounter more job-related anxiety and depression (Hegney et al., 2014; Karanikola et al., 2016) due to complex treatment and patient conditions (Nia et al., 2016; Rodrigues & Chaves, 2008). Anxiety among this specific population needs more emphasis, especially newly hired nurses in oncology settings (Karanikola et al., 2016). Newly hired nurses exposed to the oncology environment would have more anxiety and may lack proper stress coping skills (Hinds et al., 1994; Rodrigues & Chaves, 2008; Wazqar, 2019; Wazqar et al., 2017).\n\nThe nursing induction program is vital to prepare newly hired nurses to smoothly fit into a new working environment and enable them to provide safe patient care. Applications to several accreditation agencies at the national and international level are required, such as the Health Care Accreditation Council (HCAC), Joint Commission International (JCI), and Magnet accreditation. Yet, the anxiety level of newly hired nurses in the oncology setting is not studied well. Thus, the findings of this study will add valuable information about the anxiety level among newly hired oncology nurses.\n\nAnxiety among nurses is a common problem and has been studied extensively worldwide (Chen et al., 2016; Cheung et al., 2016; Creedy et al., 2017; Glazer & Gyurak, 2008; Huang et al., 2018; Wang & Zhang, 2017; Xianyu & Lambert, 2006). One study assessed the prevalence of anxiety and other mental disorders among 102 clinically active Australian nurses who work at hospitals showed that 41.2 % of nurses had anxiety (Maharaj et al., 2019). In China, a study conducted in seven governmental hospitals in one city found that the anxiety prevalence rate among Chinese nurses was around 43.4% (Gao et al., 2012). Another study was conducted in the Czech Republic, comparing anxiety prevalence between general nurses and Intensive Care Unit (ICU) nurses at three different hospitals found that 44% of general nurses had anxiety. Simultaneously, the prevalence of anxiety among ICU nurses was 28% (Janda & Jandová, 2015). Furthermore, Taghinejad et al. assessed 86 Iranian nurses working in the mental health sector at three different general hospitals; they found that 16% of nurses had an anxiety symptom (Taghinejad et al., 2014).\n\nHospital settings are a very stressful working environment for healthcare professionals, especially for front-line nurses. A study that examined nursing workplace stressors at different hospitals found that increased work demands, caring for dying patients, and conflict with other healthcare providers are common sources of nursing work-related anxiety (Jafari et al., 2018; Khodadadi et al., 2016). In another study, Janda and Jandová (2015) performed specific tasks, types of patients, and the increasing work demands were the causes of nurses' work-related anxiety. Moreover (Saquib et al., 2019), studied the anxiety and stress prevalence among non-Saudi foreign nurses associated with job dissatisfaction; the results showed that the anxiety level was significantly associated with the workload.\n\nA study was conducted to assess anxiety intensity symptoms of Greek oncology nurses using the Hamilton Anxiety Scale (HAS), and it showed that 11% of oncology nurses had a moderate intensity of anxiety symptoms (Karanikola et al., 2016). Furthermore, in Iran, at a teaching hospital, Molavynejad et al. (2019) investigated the relationship between burnout level and personality traits among 106 oncology nurses; the results revealed that 32.1% of the oncology nurses had severe burnout with a significant positive correlation with anxiety (Molavynejad et al., 2019).\n\nNewly hired oncology nurses may lack proper anxiety and stress coping skills (Hegney et al., 2014). Therefore, it is crucial to assess the anxiety level among newly hired oncology nurses and implement intervention programs to prepare them for the new working environment safely. Many studies have been conducted to explore GNO's effect on improving newly hired nurses' skills and knowledge (Cockerham et al., 2011; Woolwine et al., 2019). In one study, Cockerham et al. (2011) evaluated the average of the pretest scores was 66% and after completing the program, the average score increased to 92%. GNOs also showed that newly hired nurses were successfully integrated into the hospital team (Bahouth & Esposito-Herr, 2009).\n\nFurthermore, other researchers conducted a literature review focused on the effect of the GNO program versus the one-year residency program on nurses' retention and turnover. They found that newly-graduated nurses' satisfaction and retention were higher with the one-year residency program versus the orientation program alone (Eckerson, 2018). Nurses reported improvements in their perception of patient care quality when a senior CRN accompanied them. A quality improvement project, conducted at a labor and delivery floor in an academic medical center to assess the efficacy of the CRN role in supporting units with high percentages of new-to-practice staff, found that the perception of quality patient care increased from 16 % pre-implementation of the CRN role to 50% post-implementation (Maloney & Nelson, 2013).\n\nAnother study was conducted to assess anxiety changes among newly hired nurses at different points in time during their first year of employment; the results indicated that the anxiety level was 12.21 after one week of the residency program to 14.17 after one month and 15.30 at the end of the second month (Lin et al., 2020).\n\nThe current study aimed to assess the anxiety level among newly hired nurses in a specialized oncology hospital throughout their initial period of employment, deploying a GNO and the CRN role.\n\n\nMethods\n\nA longitudinal one-group repeated measure design was used in the study (Salkind, 2012). In a longitudinal study in which progress over time is measured, repetitive observations are gathered (Polit & Beck, 2016).\n\nThe study used purposive sampling of newly hired registered nurses, with inclusion criteria of having at least a bachelor's degree, attending the GNO, and provision of nursing care directly to patients with cancer. The purposive sample was used as we sought a selection with particular criteria that helped the study purpose (Patton, 2005).\n\nThe anticipated sample was calculated using G*-POWER with a consideration of having an alpha of .05, a power of .80, a medium effect size (d = 0.25) (Cohen, 1988), which resulted in a sample of 128 participants. Oversampling was intended to overcome the attrition rate if the participants could not be reached or if there was an incomplete questionnaire. So, a total of 200 participants were selected.\n\nThis study was conducted at a specialized oncology hospital, a tertiary cancer center in Jordan. It receives more than 3500 newly diagnosed oncology patients every year. This hospital is equipped with advanced technology, medical equipment, and services, comprising 352 beds, including two ICUs, one for adults and one specialized for pediatrics. Oncology care is provided by qualified oncologists and healthcare experts, such as trained nurses, especially in oncology care nursing, who work collaboratively to guarantee that patients receive safe and advanced cancer care.\n\nThe State-Trait Anxiety Inventory for Adults™ (STAI-AD) is a reliable tool for assessing adults' anxiety levels. The tool is translated into many languages. The internal consistency mean was .89 for the trait scale and .91 for the state scale. The test-retest reliability mean for the trait scale was .88. As expected, the state scale mean was lower than .70, indicating the transitory nature of the measure (Barnes et al., 2002). Moreover, Spielberger et al. (1999) have displayed good concurrent validity, as scores on the STAI correlate extremely with findings observed on alternative anxiety measurement tools such as the Anxiety Scale Questionnaire.\n\nFive experts assessed the content validity of the STAI-AD, determining if the items correctly reflected the STAI characteristics. A psychologist, an oncology nurse, a psychiatrist, a nurse manager, and a psychosocial specialist were among the experts. In order to determine the extent of relevancy to the newly hired nurses, experts were asked to evaluate each item in the tool. The scoring system was a scale from 1 to 4 (4 = very relevant, 3 = relevant, 2 = slightly relevant, 1 = not relevant to anxiety level among newly hired nurses).\n\nThe two STAI subscales are performed and scored from one to four. Participants apply a 4-point Likert scale for the state part to reflect how certain items about anxiety refer to the individual at a specific right moment. Likert scales range from 1 (“not at all”) to 4 (“very much so”). The trait part provides a comparable 4-point scale but directs how participants feel in general. The Likert scale for this part ranges from 1 (“almost never”) to 4 (“almost always”). Scores are calculated to give a total score for each subscale.\n\nEvery item in the STAI is given a weighted score of 1 to 4. A score of 4 shows the presence of a high anxiety level such as “I feel upset” and “I feel frightened.” Also, the scoring weights for the anxiety-negative items were reversed. We added the weighted scores for the 20 items on each scale. Therefore, scores for both the State-Anxiety and the Trait-Anxiety scales can vary from a minimum of 20 to a maximum of 80.\n\n“This instrument is covered by the U.S. and international copyright laws as well as various state and federal laws regarding data protection. In whole or in part, any use of this instrument is subject to such laws and is expressly prohibited by the copyright holder. If you would like to request permission to use or reproduce the instrument, in whole or in part, contact Mind Garden, Inc.” (Spielberger et al., 1999).\n\nThe concepts of state and trait anxiety were often used in literature; basically, personality states refer to what individual feeling and emotional reactions at this moment of time induced by stressful experimental procedures such as starting a new job. Subjective individualized feelings would reflect anxiety while personality traits indicate individuals generally feel.\n\nData collection included participants' demographics sheet and the STAI-AD. Data were collected on day one of the GNO, upon completion of GNO, and after 90 days of the intensive unit-based orientation program with clinical resource nurses (CRN). All newly hired nurses were trained to provide safe, optimal, and excellent patient care through a nursing induction program consisting of a GNO program followed by a mentorship period with a CRN. A CRN is responsible for administering clinical expertise to newly hired nurses. The CRN is qualified to monitor and help the nursing team to provide nursing care based on best practices. Clinical activities may include direct care to the patients and coordination with the healthcare team.\n\nThe nursing induction program is specially developed to prepare newly hired registered nurses to work safely and independently. It includes identifying the organization's values, mission, vision, and objectives, which would help the nurses enroll in the new environment smoothly. However, it also stresses vital patient safety issues, a standard of patient care and provides core mandatory training and competencies required to begin their new job.\n\nThe GNO program extends over ten working days and scheduled quarterly data collected from May 2018 to Aug 2019. All newly hired nurses attend a series of lectures focused on patient safety, primary nursing care, stress management, and advanced oncology care. Additionally, a three-month mentorship program with a CRN at the unit level would be completed to ensure that nurses accomplish all required competencies. Our hospital induction program was created using a competency-based assessment method, “Novice to expert clinical practice model” (Benner, 1984). The activity-based teaching/learning strategies used in the GNO program support learners' combined knowledge with proper clinical situations.\n\nEthical approval to conduct this study was received from the Institutional Review Board (IRB) at King Hussein Cancer Center. All methods were performed in accordance with the relevant guidelines and regulations. The researchers obtained an institutional review board approval to conduct the study; the approval code is 18KHCC36.\n\nThe participants were met in a private room to explain the study's goals and discuss consent to participate in the study. We explained and described the study's purposes and the research outcomes for each participant. Then, the researcher(s) asked the participants to sign the consent form. All forms were numerically coded before being given to the participants. RNs were provided envelopes to put the completed forms to ensure anonymity and confidentiality. No particular participant information could be associated with any response.\n\nThe questionnaire and the STAI-AD were administered through a paper and pencil survey. Newly hired registered nurses voluntarily filled out the survey at the beginning of the GNO and upon completion, as well as after 90 days of the GNO.\n\nStudy design, sampling, and selection were determined in the initial planning stage of a study to reduce any potential source of bias. Moreover, 19 participants were excluded from the study and analysis due to incomplete outcomes assessment.\n\nData were analyzed using Statistical Package for the Social Sciences (SPSS) version 21. Descriptive and inferential statistics were used to meet the study's aims at a significance level of .05. Descriptive statistics were used to describe demographic characteristics based on the level of measurements. One-way repeated measured analysis of variance (ANOVA) was used to compare the anxiety level at different periods of time.\n\n\nResults\n\nQuestionnaires were distributed to 200 nurses; at the beginning of the GNO, 200 participants returned the questionnaire with a response rate of (100%). Upon completion of the GNO, 188 participants returned the questionnaire with a response rate of (94%), and after three months of the CRN role, 181 returned the completed questionnaire (90.5% response rate). Demographic data showed that 64.6% of participants were female, 93.9% single nurses; most of the participants had a bachelor in nursing degree and had Jordanian nationality (n=180, 99.4%). The mean of participants' age was 23.30 years (SD=3.52), while the average of their past experience years was 1.02 (SD=1.01) (Table 1).\n\nThe study results revealed that the anxiety level among the nurses was lowest in their general daily life. However, at the beginning of the study, the mean anxiety level was 39.2 (SD=8.61); and, the mean of anxiety among participants was highest after 90 days of employment ((45.71, SD=7.20). The participants' anxiety level increased gradually from the initial contact at day one of the GNO, then on to the last day of GNO, and finally after 90 days of employment (Table 2).\n\nANOVA was used to evaluate the change in participants' level of anxiety when measured before participating in the GNO, on the last day of GNO, and after 90 days. The results of the repeated measure ANOVA revealed a significant time effect, Wilks’ Lambda=.668, F (2, 179)=44.47, p>.001, n2=.332. Follow-up comparisons revealed that each pairwise difference was significant, p>.001 (Table 3). So, there was a significant increase in anxiety levels over time.\n\na Exact statistic.\n\nb Computed using alpha=.05.\n\nThe Cronbach Alpha Reliability was .893 and .865 for STAI Y1 (Present feeling) and Y2 (General feeling), respectively (Table 4).\n\n\nDiscussion\n\nThe current study's primary objective was to assess the anxiety level among newly hired oncology nurses at three different times during the nursing induction program. A large portion of nurses included in this study were newly graduated nurses.\n\nThis study's results indicate an increase in the anxiety level among newly hired nurses in the oncology hospital from the beginning of employment to the end of the induction program. Overall, the findings revealed that newly hired oncology nurses in this study had different levels of anxiety. This finding is consistent with previous studies, which showed that nurses reported anxiety (Karanikola et al., 2016; Molavynejad et al., 2019). Compared to other nursing specialties, a study was conducted among Critical Care nurses in Greece, which showed that around a quarter of participants reported moderate to severe anxiety symptoms (Karanikola et al., 2012). In another study conducted in China, almost 43.4% of nurses experienced anxiety (Gao et al., 2012), which was also consistent with another study conducted among nurses in Iran (Kayalha et al., 2013). Other nursing studies reported a high to moderate anxiety levels among non-oncology nurses worldwide (Chen et al., 2016; Cheung et al., 2016; Creedy et al., 2017; Glazer & Gyurak, 2008; Huang et al., 2018; Wang & Zhang, 2017; Xianyu & Lambert, 2006).\n\nThe study findings indicate that newly hired oncology nurses' anxiety levels were consistently increasing from the beginning of the GNO program to the end of the GNO, and three months period subsequently. These results are similar to the findings of Lin et al. (2020); who reported that anxiety level reaches the peak after three months of employment among newly hired nurses despite enrolling them in a well-structured nursing residency program (Lin et al., 2020). This result could be viewed in the context of our nursing induction program schedule and timetable as the first ten days of the GNO program include class-based lecturing away from the clinical setting. These lectures focus primarily on general concepts related to policy, procedure, and oncology patient care; therefore, the newly hired nurses' anxiety level is low. However, as the induction program progressed, the workload of newly hired oncology nurses increased gradually to meet the orientation program expectations, which led to increasing the nurse's anxiety.\n\nOther studies also revealed that having a complex relationship with other healthcare providers was a factor causing work-related anxiety among nurses in the oncology setting (Escot et al., 2001; Isikhan et al., 2004). The direct supervision role performed by the senior CRN could contribute to increasing newly hired nurses' anxiety in the clinical setting, along with the fact that they have to pass all required competencies to become independent practitioners. Additionally, by the end of three months of employment, a probationary evaluation would increase the levels of anxiety of the newly hired nurses. Most participants were new graduate nurses, young with little previous clinical experience, which meant a lack of proper stress coping skills. Similarly, Hegney et al. in 2014 showed that anxiety levels are higher among younger nurses, a full-timer, and without specific postgraduate qualification and training (Hegney et al., 2014).\n\nAnother explanation of the current study findings that, in oncology hospitals, nurses are looking after patients who require frequent nursing care; the workload increases gradually as the program goes on, which leads to an increase in nurses' anxiety. Additionally, nurses working in oncology settings might encounter more job-related anxiety and depression due to the type of medical situations such as treatment complexity and patient conditions. Keskin et al. found that workload and an increasing number of patients lead to anxiety and depression among oncology nurses (Keskin et al., 2018). Subsequently, nurses witness the patients' suffering, family distress, and possible death at a certain disease progression stage. In such situations, newly hired oncology nurses' ability to cope with end-of-life situations influences the oncology nurse's death anxiety level.\n\nThe unavailability of a psychological support intervention for newly hired oncology nurses during the hospital induction program could explain the current study findings. The program focused on improving nurses' knowledge and skills through the oncology training course and introducing the end-of-life concept. The absence of psychological support intervention flags the importance of how to support newly hired oncology nurses once they are exposed to stressful and unpleasant situations. Several studies highlighted the importance of integrating psychological support intervention in clinical environments such as art and music therapy (Doo et al., 2018), using emotional intelligence as anxiety tool management (Kadda, 2014), mindfulness-based programs (Foureur et al., 2013), and guided imagery (Boehm & Tse, 2013).\n\nThe newly hired nurses in this study had anxiety despite completion of the nursing induction program; limited literature looked at the effect of GNO and mentorship program by CRN on newly hired nurses at the oncology center. Our results are consistent with Cockerham et al., who investigated the effect of GNO and mentorship programs on improving newly hired nurses' knowledge and skills; they found that the average of the pretest scores was 66%, and after completing the program, the average score increased to 92%. Hence Lin et al. (2020) focused on exploring changes in anxiety level and work stress among newly hired nurses over the two-year residency program. They conclude that anxiety reached its peak at the first three months of employment and then stabilized until the end of the program. The current study results provide baseline data for the further nursing study. Furthermore, the organization invests in human resource development creating clinic-oriented educator role such as CRN to facilitate unit-based orientation programs and mentorship. Thus, this would ease the new nurses' enrollment and decrease the anxiety associated with the new role and/or environment.\n\nThe current study has clinical implications and suggestions for planning interventions to assist oncology nurses in reducing levels of anxiety. Nurse leaders, managers, and educators should create new interventions such as support groups, physical and mental exercises, counseling support, and anxiety control sessions, motivating nurses to verbalize their feelings to help nurses completely control their own anxiety (Aycock, 2009; Henry, 2014). Oncology hospitals should provide healthy working environments and create particular interventions to decrease nurses' stressors. Given these results, anxiety-related work should be addressed and controlled to avoid growing tension among nurses and support nurses during the first phase of employment to reduce or eliminate anxiety.\n\nThe study was conducted at a specialized oncology hospital in Jordan. The cross-sectional design can limit the inferences of causality (Brady Germain & Cummings, 2010). Moreover, the study was limited to assessing the oncology nurses' anxiety level; the investigators didn't determine the intensity of anxiety for nurses. Given the standard procedure to conduct a GNO to newly hired nurses, assessing its impact versus not receiving a GNO (control group) without compromising patient care on anxiety levels is difficult. Further studies are recommended to evaluate the severity of anxiety for this target population more accurately.\n\n\nConclusion\n\nNurses working in oncology workplaces frequently encounter several anxiety conditions in their settings, leading to psychosocial and physical problems. So, nurses should be willing to care for themselves to maximize the optimization of their health and to decrease anxiety at work; on the other hand, seeks Nursing administrator attention to address this phenomenon and modify nursing induction programs in order to apply proper strategies to reduce anxiety level among newly hired nurses also help them to fit into a new team smoothly to ensure safe patients care.\n\n\nData availability\n\nHarvard Dataverse: “Anxiety Level among Newly Hired Nurse in a Specialized Oncology Hospital”, https://doi.org/10.7910/DVN/OSXHWE (Alloubani, 2021).\n\nThis project contains the following underlying data:\n\n- Anxity research - Statistics.tab\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\nAlloubani A: Anxiety Level among Newly Hired Nurse in a Specialized Oncology Hospital.2021. 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PubMed Abstract \| Publisher Full Text\n\nWang Y, Zhang B: Impact of personality trait and professional identity on work-related depression, anxiety and irritation among Chinese nurses. Southeast Asian J. Trop. Med. Public Health. 2017.\n\nWazqar DY: Oncology nurses' perceptions of work stress and its sources in a university-teaching hospital: A qualitative study. Nurs. Open. 2019; 6(1): 100–108. PubMed Abstract \| Publisher Full Text\n\nWazqar DY, Kerr M, Regan S, et al.: An integrative review of the influence of job strain and coping on nurses' work performance: Understanding the gaps in oncology nursing research. Int. J. Nurs. Sci. 2017; 4(4): 418–429. PubMed Abstract \| Publisher Full Text Chinese Nursing Association.\n\nWoolwine S, Romp CR, Jackson B: Game On: Evaluating the Impact of Gamification in Nursing Orientation on Motivation and Knowledge Retention. J. Nurses Prof. Dev. 2019; 35(5): 255–260. Publisher Full Text\n\nXianyu Y, Lambert VA: Investigation of the relationships among workplace stressors, ways of coping, and the mental health of Chinese head nurses. Nurs. Health Sci. 2006; 8: 147–155. Publisher Full Text" }	[ { "id": "123036", "date": "01 Mar 2022", "name": "Nazih Abu Tabar", "expertise": [ "Reviewer Expertise Nursing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this article.\nIn general, the study construction is very good. It is easy to read and follow. The study topic is discussing important factors that affect the nursing profession. The authors succeed in describing the problem and highlighted the importance of reviewing these issues in the nursing profession and among Jordanian nurses as the targeted country of the study population.\nThe methods used in the study are a longitudinal, one-group repeated measure design. A valid and reliable questionnaire was used. Given the stated aim of the study to “assess the anxiety level among newly hired nurses through three periods of employment at the specialized oncology center. However, the main worries related to the methods are:\nThe sampling method is a purposive sample, was the sample size affected by rates of refusals or attrition? if yes so it is a convenient sample method. ‎The method of calculating total sample size is not defined by which statistical test was used to decide if it is sufficient to meet the assumption of used statistical analysis test. So, was the sample size enough to support the conclusion validity? Another worry in the methodology is not clear which scale was used in the state-trait anxiety inventory questionnaire.\nThe Results of the study are clear and well presented. The statistical analysis seems to be appropriate given the study aim, variables, and nature of the data collected.\nThe Discussion seems very good, it compared the results of the current study with similar national and international literature. It suggests the implications of the results and interventions of the study to support the newly hired nurses. The critical point in the discussion, the authors try to prove the impact of the nursing induction program which highlights some concerns about the aim of the study and methodology were used. The limitations need some revision and editing because it mismatches with study aims and methodology.\nOverall, this article includes significant information in a particular setting that can be useful in guiding efforts to promote and support an appropriate intervention to support a newly hired nurse.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "127075", "date": "28 Mar 2022", "name": "Elham H. Othman", "expertise": [ "Reviewer Expertise Nursing research. Oncology and End of Life care." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this article, which addressed a significant issue among nurses working with oncology patients. The research team proficiently presented their study and findings. However, I suggest a few modifications to improve the quality and readership of the paper.\nIntroduction: The introduction section was well-written and comprehensive. However, I noticed redundancy in presenting previous literature, which can be re-phrased to improve the reading flow.\nIn light of the study aim, I suggest modifying the following paragraphs, as these paragraphs addressed the impact of the GNO or CRN on staff anxiety, which might confuse the readers about the study aim.\nNewly hired oncology nurses… hospital team (Bahouth & Esposito-Herr, 2009).\n\nFurthermore, other researchers … 50% post-implementation (Maloney & Nelson, 2013).\n\nMethodology: In the sampling section, you may specify the duration of data collection. As you mentioned, you collected data from 181 newly hired nurses, were they engaged in the same period?\nIn the instrument section: In the second paragraph: “Five experts assessed the content validity of the STAI-AD, determining if the items correctly reflected the STAI characteristics.” Is that necessary, as the instrument is already validated? And suppose you mean that it was validated to determine the extent of relevancy to the newly hired nurses. In that case, I suggest modifying the first sentence and adding the results of the experts’ validation.\nResults: You presented the Cronbach Alpha Reliability twice, in a paragraph and in Table 4, which is redundant. I suggest keeping the text and deleting the Table.\nDiscussion: You mentioned that a large portion of nurses included in this study were newly graduated nurses; it would be better if you added the number and percentage of these nurses in the result section.\nIn the sixth paragraph, you stated: ‘The newly hired nurses in this study had anxiety despite completion of the nursing induction program; limited literature looked at the effect of GNO and mentorship program by CRN on newly hired nurses at the oncology center’. This sentence implies that the study examined the effect of such programs on anxiety rather than only observing and describing the anxiety levels. I suggest modifying it according to your study aim.\nIn the same paragraph, you said: ‘Our results are consistent with Cockerham et al., who investigated the effect of GNO and mentorship programs on improving newly hired nurses' knowledge and skills’. However, I am not sure how that is consistent with your study. The cited study measured knowledge and skills, while you measured anxiety. I advise deleting or replacing it.\nIn the Limitation section, you said ‘The cross-sectional design can limit the inferences of causality,' which is irrelevant as you conducted a longitudinal study. Please correct.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-162
https://f1000research.com/articles/11-161/v1	09 Feb 22	{ "type": "Research Article", "title": "Care-seeking behaviour of adolescents with patellofemoral pain: a retrospective cohort study", "authors": [ "Michael Skovdal Rathleff", "Camilla Rams Rathleff", "Jens Lykkegaard Olesen", "Ewa M Roos", "Sten Rasmussen", "Alessandro Andreucci", "Martin Bach Jensen", "Camilla Rams Rathleff", "Jens Lykkegaard Olesen", "Ewa M Roos", "Sten Rasmussen", "Alessandro Andreucci", "Martin Bach Jensen" ], "abstract": "Aim: The aim of this study was to assess the care-seeking behaviour among adolescents with patellofemoral pain (PFP). Methods: This retrospective study included data on 121 adolescents with PFP enrolled in a randomized controlled trial. A questionnaire was sent to the general practitioner (GP) of each adolescent, assessing information on the consultation dates for knee pain, potential diagnoses, and treatment provided. Results: 106/121 adolescents had been in contact with their GP, and 95 medical records of adolescents were available. Of the 95 adolescents with available medical records 60 had consulted their GP for knee pain. The median number of contacts was 1.5 (range 1-7). The GPs initiated treatment for 48 of the 60 adolescents and in most cases it was information and advice (36/48) or pain medication to a minor extent (6/48). Out of the 60 adolescents who consulted their GP 26 were subsequently referred to different types of health care professionals, in 11 out of 26 to physiotherapy, but also to the department of rheumatology or orthopaedics. Conclusions: 63% of adolescents diagnosed with PFP had previously consulted their GP due to knee pain. Several types of treatments were initiated by the GP, but most commonly advice and information were given. Standardized and evidence-based treatment guidelines for adolescent knee pain in general practice are needed", "keywords": [ "Adolescents", "care-seeking", "knee pain", "patellofemoral pain", "treatment" ], "content": "List of abbreviations\n\nAPA2011: Adolescent Pain in Aalborg 2011\n\nGP: General Practitioner\n\nPFP: Patellofemoral pain\n\nRCT: Randomized controlled trial\n\n\nIntroduction\n\nMusculoskeletal pain is experienced by up to 40% of adolescents1–3 and is a common reason for consulting a general practitioner (GP), who is often the first healthcare professional involved in the management and treatment of pain.4–6 The knee is one of the most prevalent regions of reported pain.2,7–9 The prevalence of knee pain in adolescents is between 19 and 31%2,8,10,11 with patellofemoral pain (PFP) being one of the most common knee conditions among adolescents, experienced by approximately 6-7% of them.6,12–14 Patellofemoral pain is defined as pain in the peri- or retro-patellar area experienced during activities that involve bending of the knee (cycling, climbing stairs or similar activities). Issues associated with long-standing PFP include high pain intensity and low quality of life. Long standing PFP is also associated with higher risk of interrupting participation in sport activities.12,15\n\nThere are a number of different treatment options for PFP. A meta-analysis from high quality trials documents a positive effect of exercise therapy.16 Exercise therapy has been advocated as the cornerstone in treatment of patients with PFP.16 A randomized controlled trial (RCT) showed that exercise therapy combined with patient education was more effective compared to patient education alone.13,17 At 12 months only 29% were fully recovered in the randomised group with patient education alone while 38% were fully recovered in the randomised group with patient education combined with exercise therapy. The proportion of adolescents fully recovered was significantly lower than what have been reported in previous similar exercise studies on adults with PFP (62-81% of patients receiving exercise therapy).18,19\n\nOn average, the adolescents in the RCT reported knee symptoms for more than three years at inclusion, which is longer than previous trials in adult patients with PFP.13,18–20 A long symptom duration before initiation of treatment is associated with poorer outcome after treatment which may partially explain the lower effect of exercise therapy among adolescents.20 The question is if these adolescents had previously contacted their GP due to their knee pain, and if so, which recommendation was provided by the GP. This is important as early treatment may improve the effect of exercise therapy among adolescents with PFP,21,22 and exercise therapy can only be prescribed if the adolescents decide to seek medical care as the GP is gatekeeper in Denmark. The purpose of this study was therefore to retrospectively assess the care-seeking behaviour of a cohort of adolescents with PFP using patient records from their GP.\n\n\nMethods\n\nThe design was a retrospective cohort study.13 Ethical approval was obtained from the local ethics committee in the North Denmark Region (N-20110020) and the ethics committee approved that adolescents >15 years of age could consent on their own. All participants were required to give written informed consent for the researchers to use their data to explore the care pathway.\n\nAdolescents with PFP were recruited from a population-based cohort (Adolescent Pain in Aalborg (APA) 2011, the APA2011-cohort) which included 2200 adolescents between 15-19 years.17 Of the 2200 adolescents who answered the questionnaire, 724 reported knee pain. A total of 504 adolescents were successfully contacted and asked standardised questions in a telephone interview.6 If they reported anterior knee pain with an non-traumatic onset, as opposed to traumatic onset, they were offered a clinical examination at the local hospital by an experienced rheumatologist to determine the specific knee condition. Two hundred and four adolescents were invited for a clinical examination and 180 accepted, 8 adolescents did not show up, leaving 172 adolescents who were examined. Of these, 153 were diagnosed with PFP, but 32 were subsequently excluded with the main reasons being the worst pain experienced the previous week being less than 30 mm on visual analogue scale (VAS) and patients with additional knee conditions (e.g. PFP combined with iliotibial band syndrome). Of the remaining 121 adolescents there were 106 who consented to contacting their GP (n=55 in total) and assess information regarding the period with patellofemoral pain prior to being enrolled in the APA-2011-cohort and a subsequent RCT.13 These 106 were the focus of the current report and these data has not been published before or used in other reports.\n\nFive GP’s never responded to our enquiry. Questionnaires containing information for 98 adolescents from their medical record were received from the remaining 50 GPs. The medical records were retrieved using the adolescent’s unique personal identification number. Of these, 3/98 adolescents had shifted to the current clinic shortly before the questionnaire was sent and it was not possible for the GP to obtain the record from the previous GP. Therefore, a total of 95 adolescents were included in the data analyses.\n\nTo obtain information from the GP, a questionnaire was developed and pilot-tested to ensure comprehensibility. The first version of the questionnaire was distributed among three GPs. The GPs were asked about comprehensibility and if there were questions that may be misinterpreted. After their feedback, a second version of the questionnaire was implemented and tested among two other GPs. Thereafter the questionnaire37 was deemed comprehensible with a minimal risk of non-comprehension and misconception.\n\nContent of the questionnaire:\n\n• The dates for all primary consultations regarding knee pain\n\n• If there were more than one contact regarding knee pain, did the record suggest it was about the same type of knee pain\n\n• Localisation of knee pain\n\n• Physical examination - observations\n\n• Diagnosis\n\n• If treatment was initiated and what type of treatment\n\n• If the patient was referred to another health professional\n\nTo make it easier for the GP to respond, each question was followed by pre-defined response options. The question on the diagnosis for example contained the following response options: Patellofemoral pain, patella tendinopathy, fat pad impingement syndrome, Mb. Osgood Schlatter, iliotibial band syndrome, and other diagnoses. If they chose “other diagnoses” they were asked to describe the diagnosis in free text. The questionnaire is available online as extended data.37\n\nDemographics are presented as mean and standard deviation except for non-normally distributed data, which are presented as median and interquartile range. Number of contacts, diagnosis, treatments given, and referrals were described as n/total n and 95% confidence interval for the proportion. No statistical hypothesis testing was done.\n\n\nResults\n\nThe majority of the sample consisted of females (81%) with a median age of 17 (Table 1) and a pain duration of more than three years (Table 1).\n\n* Median (interquartile range).\n\nThe questionnaires based on the GPs medical record showed that 60/95 (0.63, 95%CI: 0.53-0.72) of the adolescents had sought medical care for their knee pain and 30/60 (0.50, 95%CI: 0.38-0.62) had consulted their GP more than once because of knee pain, Figure 1. Among those who consulted their GP more than once, the GP suspected the same knee condition in 8/30 (0.27, 95%CI: 0.14-0.45) adolescents. The median number of contacts was 1.5 (range 1-7).\n\nThere was good agreement between the information provided by adolescents regarding prior consultations and the information retrieved from the medical records at the GP. From the 60 adolescents who had consulted their GP because of knee pain, 50/60 (0.83, 95%CI: 0.72-0.91) reported having consulted their GP because of knee pain.\n\n26/60 (0.43, 95%CI: 0.32-0.56) of the adolescents who consulted their GP were subsequently referred. In Figure 1 the referrals are described (bottom line). Most frequently, they were referred to physiotherapy (n=11).\n\nIn 12/60 cases (0.20, 95%CI: 0.12-0.32) (20%) no treatment was initiated by the GP, and from these, eight were later referred. The GP initiated treatment in 48/60 (0.80, 95%CI: 0.68-0.88) adolescents who contacted the GP. The most common recommendation provided was oral information and advice (36/48) (0.75, 95%CI: 0.61-0.85) followed by pain medication (6/48) (0.13, 95%CI: 0.05-0.30).\n\nData on diagnosis was obtained for 51 adolescents, Table 2 and Figure 2.\n\n20 adolescents had pain isolated to the anterior aspect of the knee, 6 had medial knee pain, 5 had lateral knee pain, 11 had a combination of anterior and medial/lateral/posterior knee pain (not shown), 3 had posterior knee pain, 5 had diffuse knee pain without a specific location. In 10 of the adolescents there were no data on pain location available.\n\nThe most common objective findings were pain at palpation and swelling, Table 3.\n\n\nDiscussion\n\nThis study showed that less than 2/3 of the adolescents with PFP consulted their GP because of knee pain. Among adolescents who consulted their GP, treatment was initiated by the GP in 48/60, while 26/60 at a later consultation were referred to primary or secondary care. These findings demonstrate the need for initiatives addressing both the adolescents and the GPs to ensure early standardised and evidence-based treatment of adolescent PFP.\n\nThe percentage of adolescents who consulted their GP because of knee pain is similar to patients with generalised pain. Roth-Isiqkeit et al. (2005) reported that 51% of children and adolescents with pain had consulted their physician23 while Perquin et al. (2000) reported that 57% had consulted their GP.24 Masiero et al. (2010) reported that 74.2% of adolescents with musculoskeletal pain consulted a health provider, which in the majority of cases was a primary care practitioner, paediatrician or orthopaedist.25 In the present study, the percentage (63%) who consulted their GP because of PFP pain as assessed by medical records is similar to our previous work on 504 adolescents with both traumatic and non-traumatic onset knee pain, where data were self-reported by participants (59%).6 Previous studies reported that high pain intensity, long pain duration and older age were associated with seeking medical care among adolescents.6,24 This is in line with the characteristics of our sample where the average age was 17, the average pain during activity was 49.5 out of 100 on the VAS and the average pain duration was 37.5 months. Therefore, an intriguing question is why so few adolescents consulted their GP? As outlined in the “Iceberg Theory of Disease”, not all people with medical symptoms decide to consult their GP.26,27 There are several factors affecting the decision to consult the GP. These include the familial patterns of consultation (especially the mother providing advice for self-treatment), advice from friends, knowledge about the condition and psychological factors (i.e. stress, perceived vulnerability to illness, perceived severity of symptoms, perceived costs and benefits of action).26–28 Previous studies also showed that the most common aetiology associated with seeking medical care for musculoskeletal pain was trauma.5,6 Conversely, all adolescents with PFP in this study reported a non-traumatic onset of knee pain. It may be that adolescents with a non-traumatic onset of knee pain, which is a gradually evolving condition, are less likely to take their knee pain seriously or to act on their knee pain as opposed to those whose knee pain originates after a traumatic injury.6 In addition, a subgroup of adolescents might decide to self-manage their knee pain with pain medication without consulting the GP.26 In adolescents, higher use of pain medication might be associated with higher age and female gender,29 in line with the demographic characteristics of our sample (mostly females in late adolescence). This is supported by our data, showing that 21% of adolescents used pain medication for their knee pain,13 and also by figures of previous studies.23,30 The tendency for self-medication behaviour is a potential issue, as evidence shows that adolescents have little knowledge on how pain medication works, potential side effects and often use pain medication inappropriately.31–33\n\nHalf of the adolescents, who initially consulted their GP, did not return for a second consultation. This could mean that their knee pain resolved after the treatment initiated by the GP. However, when the APA-cohort was initiated in September 2011, all 95 adolescents in the study had severe and long-lasting knee pain, which suggests that the knee pain did not resolve completely. The question is why the adolescents did not return to their GP and if they still had knee pain? One explanation is that they did not feel their knee pain was severe enough for the GP to be consulted and did not feel worried or anxious about their condition.26 Another possibility is that the treatment initiated by the GP was unsuccessful (or the GP recommended to “wait and see”) and adolescents did not feel confidence in the GPs ability to handle their knee pain.26 However, our data are in line with those of a recent primary care study which showed that only 45% of patients had two or more consultations and only 27.1% of patients with a persistent pain condition consulted 4 or more times.34\n\nInformation on the GPs diagnosis illustrate the variety in medical diagnoses the adolescents received the first time they consulted their GP. Quite surprisingly only 18 out of 51 adolescents were diagnosed with PFP. The rest of the medical diagnoses were a combination of tendon related pain, Mb. Osgood Schlatter and iliotibial band syndrome. The pain localisation noted in the GPs medical records was in most cases compatible with PFP, however in several patients the pain localisation and physical findings points towards other knee conditions. This suggests that previous contacts to their GP represent separate knee conditions that may be precursors to PFP. Another possible explanation is that the quality of the diagnosis provided by the GP is too low to infer if their knee pain initially started out as a different knee condition than PFP.\n\nTo our knowledge, no one has previously investigated which treatments are most commonly used to treat adolescent PFP in primary and secondary care. Our previous study on self-reported treatment showed that of 504 adolescents with traumatic or non-traumatic onset knee pain, only 18% were currently receiving treatment and the most commonly received treatments were exercises and orthotics.6 Among the 95 adolescents with PFP in the current study only 12 were referred to physiotherapy, which is the most common health professional to treat with exercises and orthotics. However, during the time period of 2006-2011 when our data was originally collected, only a few randomised trials existed and there was only weak evidence on the effect of exercises compared to information and advice. This, taken together with the variety of diagnosis given, suggests that adolescents were not treated in a standardized way at that time-point. Our data showed that 20% of adolescents (12/60) who consulted their GP were not initially given a treatment. This suggests that there is a small but considerable proportion of adolescents who are just initially told to “wait and see”. This recommendation might influence how adolescents interpret their symptoms (i.e. not severe enough for a treatment) and might affect their willingness to self-care as well as to seek treatment in case of their knee pain becoming more severe. Further studies in this area are needed to shed more light on this issue.\n\nThe results from the current study may not be generalizable to all adolescents with PFP. As we enrolled adolescents who reported knee pain in September 2011, we did not include those who had been successfully treated by their GP for their knee pain. The retrospective cohort design is limited by the amount and quality of the information the GPs noted in the medical records.35 However, because of the retrospective design the diagnosis, the choice of treatment and referral is unbiased as the GPs were not aware that their data would later be used for research purposes.36 The sample size was fixed in advance, as the adolescents with PFP were participants in a randomized trial. The participants were, however, recruited from a population-based cohort. Larger studies may provide more precise estimates and be more generalizable.\n\nThis sample included adolescents from a population-based cohort who were all diagnosed with PFP by an experienced rheumatologist in September 2011. One of the strengths of the study is that Danish citizens have free and unlimited access to health care through a GP. Therefore, the care-seeking behaviour is not biased due to unequal access to health care.36 However, our findings may not be generalizable to countries with unequal access to health care.37 In addition, patients referred to physiotherapists have to pay about €20-30 per consultation. Therefore, economic considerations might have influenced whether or not patients wished to be referred to physiotherapy.\n\nAdolescent PFP pain does not always have a favourable long-term prognosis.38 A longer pain duration before initiation of treatment is associated with poorer long-term prognosis among patients with PFP20 and early treatment is recommended compared to a “wait-and-see” approach.39 There was a large heterogeneity in the clinical pathway and the types of treatments initiated in the sample of this current study, whose data originated from 10 years ago. Since that time, however, the Danish clinical guidelines for the treatment of adolescent knee pain have not been updated. Therefore, our study suggests that there is a need to develop clinical practice guidelines for treatment of adolescent knee pain as a first step to ensure evidence-based treatment which would improve the long-term prognosis.\n\n\nConclusion\n\n60 of the 95 adolescents diagnosed with PFP had previously consulted their GP because of knee pain. There was large heterogeneity among the treatment recommendations provided by the GP, the most common being general advice and information. However, there was also a substantial proportion (20%) of adolescents who were not initially provided a treatment. These findings demonstrate the need for initiatives addressing the GPs to ensure evidence-based treatment of adolescent PFP. As a first step, these initiatives should aim at establishing clinical practice guidelines for treatment of adolescent PFP.\n\n\nData availability\n\nHarvard Dataverse: [Data regarding adolescents with patellofemoral pain] https://doi.org/10.7910/DVN/GW3JKL38\n\nThe project contains the following underlying data:\n\n- Clinical pathway data_anonymized_only english.xls (raw anonymized data from questionnaires)\n\nHarvard Dataverse: [Data regarding adolescents with patellofemoral pain] https://doi.org/10.7910/DVN/GW3JKL38\n\nThis project contains the following extended data:\n\n- Questionnaire used for data collection.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\nSoftware availability\n\nThere was no specific statistical code used for the descriptive analysis.\n\n\nAuthors' contributions\n\nAll authors contributed to the development of the study purpose. MSR, CRR and MBJ collected data. All authors interpreted the results together. MSR, CRR and MBJ wrote the first draft of the manuscript and EMR, JLO, SR and AA gave feedback and helped revise the manuscript.\n\n\nEthics approval\n\nEthical approval was obtained from the local ethics committee in the North Denmark Region (N-20110020).\n\n\nConsent to participate\n\nAll participants were required to give written informed consent for participation in the study.\n\n\nConsent for publication\n\nAll participants were required to give consent for publication together with the informed consent for participation in the study.", "appendix": "References\n\nKing S, Chambers CT, Huguet A, et al.: The epidemiology of chronic pain in children and adolescents revisited: a systematic review. Pain. 2011; 152: 2729–2738. PubMed Abstract \| Publisher Full Text\n\nRathleff MS, Roos EM, Olesen JL, et al.: High prevalence of daily and multi-site pain - a cross-sectional population-based study among 3000 Danish adolescents. 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Eur. J. Pediatr. 2000; 159: 608–614. Publisher Full Text\n\nBondesson E, Olofsson T, Caverius U, et al.: Consultation prevalence among children, adolescents and young adults with pain conditions: A description of age- and gender differences. Eur. J. Pain. 2020; 24: 649–658. PubMed Abstract \| Publisher Full Text\n\nMuller S: Electronic medical records: The way forward for primary care research?. Fam. Pract. 2014; 31: 127–129. PubMed Abstract \| Publisher Full Text\n\nDelgado-Rodriguez M, Llorca J: Bias. J. Epidemiol. Community Health. 2004; 58: 635–641. PubMed Abstract \| Publisher Full Text\n\nKringos D, Boerma W, Bourgueil Y, et al.: The strength of primary care in Europe: An international comparative study. Br. J. Gen. Pract. 2013; 63: e742–e750. Publisher Full Text\n\nNimon G, Murray D, Sandow M, et al.: Natural history of anterior knee pain: a 14- to 20-year follow-up of nonoperative management. J. Pediatr. Orthop. 1998; 18: 118–122. PubMed Abstract \| Publisher Full Text\n\nMills K, Blanch P, Dev P, et al.: A randomised control trial of short term efficacy of in-shoe foot orthoses compared with a wait and see policy for anterior knee pain and the role of foot mobility. Br. J. Sports Med. 2012; 46: 247–252. Publisher Full Text" }	[ { "id": "171007", "date": "15 May 2023", "name": "Mitchell C. Selhorst", "expertise": [ "Reviewer Expertise Physical Therapy rehabilitation of adolescents with Patellofemoral pain", "and other overuse injuries" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a clear and well written manuscript. This article was on the care seeking nature of adolescents with PFP. This was a retrospective assessment of a PFP cohort. GP's of Adolescents with PFP who were previously seen were surveyed and a review of their medical chart was completed. A little over half were given education and advice by the GP. Most adolescents did not receive a referral for further care for their PFP. Based on the current understanding of PFP, a wait and see approach may not be best and a revamp of current AKP guidelines may be necessary.\nSpecific questions and comments.\nTable 1, it may be useful to readers to include the % of adolescents for B knee pain, previously treated and medication. I.e. B knee pain 75 (79%). Similarly for gender provide the n before the %.\n\nPlease clarify those “adolescents that did not contact their GP about their knee pain”. Does this mean that they ended up in your larger research cohort without the GPs knowledge of knee pain, or does this mean that knee pain was reported on a routine healthy checkup with the GP? Often we see adolescents who reported pain at the wellcheck with GP but did not specifically contact the GP for that pain.\n\nIn the section regarding Recommendation provided by the GP. In 12/60 cases no treatment was initiated by GP….eight were later referred. Does this mean that no treatment was initiated initially and subsequently the GP referred after pain continued, or that patient was referred on by another provider?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "180243", "date": "03 Aug 2023", "name": "Alicia Fernandez-Fernandez", "expertise": [ "Reviewer Expertise Pediatric physical therapy", "biomechanics", "clinical decision-making" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written manuscript that explores care provided to adolescents with PFP, including patterns of GP consultation as well as subsequent referrals and interventions. The findings confirm that there is a need for standardization of care protocols for adolescent knee pain in general practice, given that there was inherent variability in seeking care as well as in the subsequent care received.\nI have a few comments:\nIn the \"seeking medical care section\", this sentence is unclear: \"From the 60 adolescents who had consulted their GP because of knee pain, 50/60 (0.83, 95%CI: 0.72-0.91) reported having consulted their GP because of knee pain.\" Note that the first part says 60 consulted the GP because of knee pain, but the second part says 50 did the same. Is there a typo or is the sentence just confusing?\n\nWas there any open-ended exploration of why in several cases no treatment was initiated by the GP, i.e. do you have any insight into the rationale for these choices? Based on physical findings, level of pain, other aspects?\n\nOne of the limitations that you recognize is that original data collection was a long time ago - some (or all) of the data seems to be over 10 years old. How outdated do you think your findings are in terms of standardization of GP practices for adolescent knee pain?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": [] } ]	1	https://f1000research.com/articles/11-161
https://f1000research.com/articles/11-160/v1	09 Feb 22	{ "type": "Software Tool Article", "title": "Lessons learned in virulence factor identification and data management from a hackathon on microbial virulence", "authors": [ "Brett E. Pickett", "Ryan Connor", "Tamiru Berhanu-Denka", "Sherry Bhalla", "Vyacheslav Brover", "Michael James Chambers", "Kumardeep Chaudhary", "Ousmane H. Cissé", "Allissa Dillman", "Moamen M. Elmassry", "Michael Feldgarden", "Eric Holloway", "Xin Huang", "William Klimke", "Catarina Inês Mendes", "S. Elizabeth Norred", "Jonathan Parkinson", "Samantha Sevilla", "Monica Garcia Solache", "Defne Surujon", "Udana Torian", "Vadim Zalunin", "Ben Busby", "Brett E. Pickett", "Ryan Connor", "Tamiru Berhanu-Denka", "Sherry Bhalla", "Vyacheslav Brover", "Michael James Chambers", "Kumardeep Chaudhary", "Ousmane H. Cissé", "Allissa Dillman", "Moamen M. Elmassry", "Michael Feldgarden", "Eric Holloway", "Xin Huang", "William Klimke", "Catarina Inês Mendes", "S. Elizabeth Norred", "Jonathan Parkinson", "Samantha Sevilla", "Monica Garcia Solache", "Defne Surujon", "Udana Torian", "Vadim Zalunin" ], "abstract": "Virulence is a complex mix of microbial traits and host susceptibility that could ultimately lead to disease. The increased prevalence of multidrug resistant infections complicates treatment options, augmenting the need for developing robust computational methods and pipelines that enable researchers and clinicians to rapidly identify the underlying mechanism(s) of virulence in any given sample/isolate. Consequently, the National Center for Biotechnology and Information at the National Institutes of Health hosted an in-person hackathon in Bethesda, Maryland during July 2019 to assist with developing cloud-based methods to reduce reliance on local computational infrastructure. Groups of attendees were assigned tasks that are relevant to identifying relevant tools, constructing pipelines capable of identifying microbial virulence factors, and managing the associated data and metadata. Specifically, the assigned tasks consisted of the following: data indexing, metabolic functions, virulence factors, antimicrobial resistance, mobile elements in enterococci, and metatranscriptomics. The cloud-based framework established by this hackathon can be augmented and built upon by the research community to aid in the rapid identification of microbial virulence factors.", "keywords": [ "enterococcus", "transposons", "plasmids", "antimicrobial resistance", "blast", "data indexing", "bioinformatics", "data mining", "metadata", "metagenomics", "algorithmic information" ], "content": "Introduction\n\nA variety of publications have reported the diverse mechanisms of microbial virulence (Leshem et al. 2020; Tarsillo and Priefer 2020; Nogueira et al. 2019; Saeki et al. 2020; Lange et al. 2019; Saiardi et al. 2018; Russo and Marr 2019; Feßler et al. 2018; Geisinger and Isberg 2017; Schroeder et al. 2017; Diard and Hardt 2017). In the vast majority of cases, one or more features/biomarkers can be identified and compared against databases of known virulence factors. Examples of such features can include genetic (e.g. pathogenicity islands, antimicrobial resistance (AMR) genes, mobile elements) (Kaushik et al. 2018; Vestergaard et al. 2019; Madec et al. 2017) and metabolomic (e.g. carbohydrates, lipids, small molecules) (Jia et al. 2019; Lloyd-Price et al. 2019) features.\n\nThe National Center for Biotechnology and Information (NCBI) at the National Institutes of Health hosts in-person hackathons to crowd-source the rapid development and prototyping of various methods (Connor et al. 2019). Recently (July 2019), a hackathon was convened to develop methods to detect various aspects of microbial virulence. The pipelines and methods that were generated during this hackathon were made publicly available to rapidly disseminate these tools and to enable the rapid identification of various microbial virulence factors.\n\nThe aim of this study was to develop computational pipelines that enabled cloud-based analyses to identify markers of microbial virulence in a modular and publicly available way. Attendees focused on implementing proof-of-concept strategies and methods to address “high-priority” needs. These tasks included data indexing, metabolic functions, virulence factors, AMR, mobile element detection in enterococci, and metatranscriptomics. Participants were grouped into teams, with each team developing at least one automated pipeline for their assigned task. These pipelines and workflows can be adapted by the research community to enable improved detection of various microbial virulence factors in a variety of data types.\n\n\nMethods\n\nAll data and metadata from NCBI Sequence Read Archive (SRA) were assembled into contigs using SKESA version 2.3.0 (Souvorov et al. 2018). Virulence factors, AMR genes, and mobile elements were then identified and annotated in these contigs with AMRfinder (Feldgarden et al. 2019). These data and metadata were then uploaded to storage buckets and Big Query (respectively) in the Google Cloud Platform. A table containing pointers to the above data and metadata was then generated.\n\nA script was then used to: 1) concatenate the metadata for each SRR in a subset of SRA experiments, 2) concatenate all assembled contigs for each SRR in the same subset of SRA experiments, and 3) apply a previously reported quantitative clustering by compression method (Cilibrasi and Vitanyi 2005), to identify any relationship between the complexity in contig sequence data and the associated metadata.\n\nBioProjects within the NCBI-hosted Sequence Read Archive were selected for analysis based upon the presence of shotgun sequencing metagenomic samples from the gut microbiome and paired labels identifying the sample with a health status. Disease statuses encompass individuals diagnosed with colorectal cancer, diabetes, obesity, Parkinson’s disease, and ulcerative colitis. Metagenomic gut samples from healthy individuals were also selected. A subset of 100 representative SRA accessions (Table 1) were selected for an initial characterization to prototype the metabolic functions workflow. Sequence read files for the selected SRA accessions were downloaded using SRA Toolkit version 2.9.6, followed by a read quality filter using PRINSEQ++ version 1.2 (Schmieder and Edwards 2011). All filtered reads contained paired ends, which were concatenated into a single read file using a custom script before being passed as input into HUMAnN2 version 2.8.1 using the UniRef90 full database to optimize the identification of uncharacterized proteins (Franzosa et al. 2018). Following the HUMAnN2 workflow, output files were normalized based on relative abundance followed by joining all sample tables to create a single table for gene families, path coverage, and path abundance. All three tables were preprocessed using a custom R script before being passed through a random forest classifier. A total of 100 trees were generated for the binary classification using the random forest algorithm implemented in the Python scikit-learn library. Owing to the low sample size with 18 healthy and 41 diseased samples (belonging to Parkinson's disease, obesity etc.), leave-one out cross validation was used.\n\nExperimentally validated virulence factor (VF) genes from the Virulence Factor Database (VFDB) were used to represent virulence-associated genes (Chen et al. 2016). Example metagenomes used for testing were drawn from public datasets listed on NCBI SRA and included healthy and disease-state human skin metagenomic samples (Table 1). Specifically, diseased metagenomes were drawn from the Diabetic Foot Ulcer metagenome study (BioProject: PRJNA506988) and healthy foot skin metagenomes were drawn from BioProject: PRJEB30094. Metagenomes were assembled using MetaSPADES version 3.13.1 (Nurk et al. 2017).\n\nA Hidden Markov Model (HMM) was applied to the VFDB genes to create virulence profiles. Genes were selected for which at least five different bacterial species were available. Multiple sequence alignments were generated using MUSCLE (Edgar 2004) and HMMs using HMMER3 (Eddy 2011). Genomes and/or corresponding protein coding sequences were screened with HMMSEARCH using pre-computed significance scores. Scores were calculated as 80% of the envelope alignment score of a representative sequence corresponding to its HMM. Alignments were filtered by custom scripts to extract putative virulence factors’ loci. VF sequences were concatenated, aligned and used as input for phylogenetic analyses. Phylogenetic trees were constructed using RAXML-ng (Kozlov et al. 2019) and analyzed using the R package Ape (Paradis and Schliep 2019) and Newick Utilities (Junier and Zdobnov 2010). Virulence tags were assigned based on the number of virulence loci found and phylogenetic classification. All analyses are described within a Snakemake workflow (Köster and Rahmann 2018).\n\nA support vector machine (SVM) model was also developed to classify virulent and non-virulent gene segments by training on a reference set of labelled pathogen and commensal genomes. The pathogen genomes were acquired from an NCBI Assembly search and included the species identified in the VFDB dataset. Commensal genomes were also acquired from an NCBI Assembly search, and included species selected from the NHSN Common Commensals List and from prior work (Busby et al. 2013). To characterize the novel virulence factors, the known virulence factors were masked from pathogenic genomes and non-pathogenic genomes.\n\nUtilizing FASTQ files, either by providing them directly or by a list of SRA run accession numbers, this Nextflow pipeline creates a curated list of AMR genes present in a metagenomic sample and includes their context by performing a guided assembly with the detected genes. In this case, the sample was from a preterm infant (ERR1600439) with information of the context of the detected gene. After quality control, three modes to detect AMR genes are available: “magicblast, “hmm” and “mash”, all utilizing the AMRFinder AMR database (Feldgarden et al. 2019). The first mode utilizes magicBLAST to align the sequencing data to the reference database and, after filtering by coverage breadth and depth, outputs the selected AMR genes found in the sample. The “hmm” mode takes the sequencing data and translates it into the six possible reading frames, and breaks each into open reading frames (ORFs). These ORFs are used as input to the HMMER algorithm to search against the profile HMMs from AMRFinder and selects AMR sequences based on a threshold bit score. The final mode, “mash”, builds a MASH sketch with the same AMR database and screens the reads against the sketch. This produces k-mer distances between the read set and each AMR, which then is used to extract only the AMRs that are close to the readset. The resulting AMRs, from any mode of the pipeline, are used as bait to perform a guided assembly with SKESA version 2.3.0.\n\nAll 15,000 available E. faecalis and E. faecium sequence runs were downloaded from the SRA database. Short reads were assembled using SKESA version 2.3.0 (Souvorov et al. 2018), with AMRFinder used to identify resistance genes (Feldgarden et al. 2019). Genomes were annotated using the Prokaryotic Genome Annotation Pipeline version 4.8 (Tatusova et al. 2016). BLAST (Altschul et al. 1990) was used to compare the assembled sequences against a database of transposon and plasmid sequences. Annotations were also searched for terms associated with mobile elements, including transposase, recombinase, integrase. After performing the described BLAST analyses, a report that combined and summarized these results was constructed.\n\nProcessed data was downloaded from the Inflammatory Bowel Disease Multi'omics Database (IBDMDB; PRJNA398089). Patient metadata, including inflammatory bowel disease information, was also obtained from the same source. Metagenomics and metatranscriptomics data were obtained as taxon and pathway abundance tables, respectively. Comparison of metagenomic and metatranscriptomic abundances were done using custom code.\n\nTo see whether metatranscriptomic data is predictive of disease state of an individual, seven machine learning classification models were trained and subsequently combined in a consensus model that classifies disease outcome. A random selection of a third of the individuals were placed in a test set to evaluate the performance of the consensus model and these individuals were not used in any training step. The input features consisted of the relative abundance of each metabolic pathway, which were normalized to have 0 mean and equal variance. The models trained include Decision Tree, K-Nearest Neighbor, Support Vector Machine, Logistic Regression, Random Forest, and Multi-Layered Perceptron. Each model was trained on the training data, using hyper-parameter tuning with five-fold cross-validation. The seven models were then combined using a majority vote scheme into a consensus model. The performance of the consensus was evaluated on the training and previously unseen test datasets. All machine learning analysis was done using the Python library scikit-learn.\n\nThe Github code repository for each of the described projects contains sufficient documentation to install all of the tools required for each of the workflows (see Software availability).\n\nAll workflows were designed to run on modern cloud-based hardware, such as the Google Cloud Platform. This approach can maximize data processing efforts, minimize data transfer, and efficiently complete the analyses. Overviews of each workflow are provided in Figures 1, 3, 5, and 6.\n\n\nResults\n\nBefore beginning the hackathon, we assembled the relevant sequence data from SRA into contigs prior to annotating them with known virulence factors, AMR genes, and mobile elements (for Enterococcus-related samples). We then added these processed data, together with the original SRA metadata and the pre-computed virulence metadata into the Google Cloud Platform. These data and metadata formed the basis for the downstream analyses described below.\n\nThe data indexing group focused on the underlying requirement by funding agencies to submit the experimental data, together with the associated metadata, to public data repositories. These large repositories include an extremely large corpus from which to perform data mining activities on existing data. Such activities are enhanced when the submissions conform to the findable, accessible, interoperable, and reusable (FAIR) data principles (Reiser et al. 2018). While the repositories provide a search interface that enables filtering by user-specified criteria, searching on relationships between datasets and supporting downstream data mining activities are often not supported. As such, the data included in this hackathon were all hosted in the cloud, using Big Query within the Google Cloud Platform. This approach facilitates the rapid identification of relevant datasets based on not only SRA metadata, but also using preprocessed data and metadata including assembled contigs, presence of AMR genes, and other elements in the contigs. Providing community access to this, and other relevant information in the cloud will improve the pace of developing solutions for human health.\n\nThe aim of this effort was to measure the feasibility of storing the assembled contigs, original SRA metadata, and additional metadata in a cloud-based platform to facilitate downstream analytical workflows (Figure 1). The benefit of this design is that both the tools and the data are co-hosted in the same cloud environment. This approach minimizes time required for file transfer and ensures that workflows are consistently accessible to all users.\n\nA graphical representation showing the important relationship between data generation, data analysis, and data indexing.\n\nIn addition to the storage of data, we hypothesized that the complexity, or sequence diversity, of contigs could correlate with the complexity of the associated metadata (e.g. disease state, year, host species, AMR gene presence). To quantitatively compare metadata and sequence data we used a technique based on algorithmic information theory. Briefly, this approach takes advantage of the fact that two related sequences show a high degree of sequence similarity (i.e. low genetic distance), and the files containing this information could therefore be compressed more efficiently. Similarly, files containing metadata that is highly repetitive would also compress much more efficiently. As such, we applied a previously published metric of mutual information that compares how well sequences and metadata compress individually and how well they compress when concatenated together (Cilibrasi and Vitanyi 2005) (Figure 2). This algorithm was shown to be agnostic to the input data and consequently robust in a variety of applications including DNA sequences. Their previous study showed that known biological taxonomies could be replicated without having to adjust the metric. A related paper demonstrated that comparing the dictionary sequences compared to the logarithmic size of the dictionary, it is possible to identify statistically significant sequences by length above a threshold (Milosavljević and Jurka 1993). Applying this method to pairs of SRR identifiers from SRA and their associated metadata revealed better compression of the metadata overall than the sequence data. We expect that the combination of more detailed and descriptive metadata for future SRA submissions, together with generating additional metadata from the relevant sequence data will improve the performance of this analytical method.\n\nThe computational workflow that was used to test whether sequence complexity in contigs relates to metadata complexity.\n\nThe metabolic functions group focused on identifying metabolic signatures of relevant virulence factors from a subset of the approximately one million metagenomic datasets that have been deposited at NCBI in the past two decades. Tens of thousands of these sequences were human-related. Due to the diversity in the metabolic capacity of the human microbiome, the role of the gut microbiome has been extensively studied in metabolic disorders and chronic diseases. At the same time, the value of machine learning has been recognized and applied to study complex datasets such as next-generation sequencing datasets. The goal of this effort was to uncover the missing metabolic functions that could exacerbate disease or disorder conditions. Finally, the established model would help predict the gut microbiome metabolic profile that may be associated with dysbiosis in the human body.\n\nThe goal of this project was to identify unique metabolic functions in gut metagenomes for disease states such as colorectal cancer, diabetic, obese, Parkinson's disease, and ulcerative colitis populations. We began by retrieving a variety of publicly available human samples from the SRA. These samples were derived from patients with a variety of disease states and were subjected to a novel computational workflow that transforms metagenomics sequencing reads into metabolic profiles (Figure 3). Specifically, we generated 100 trees in the random forest to select the features with the depth of 2 for pruning and classification. We then calculated the useful performance metrics for this approach including precision (1.00, 0.72), recall (0.11, 1.00), and F1-score (0.20, 0.84) for non-disease vs disease samples respectively, along with the area under the curve value of 0.86 (Figure 4). Overall, we observe that this cloud-based workflow shows a high amount of promise for differentiating between disease and non-disease samples using only metagenomic data.\n\nA graphical description of the computational workflow used to identify metabolic functions from metagenomics data.\n\nAUC quantifies the ability of the machine-learning based process to correctly assign healthy and unhealthy patients based on metagenomics data.\n\nThe virulence factors group developed methods to better understand how disease progression could be impacted by: metagenomic identification of microbial virulence, community relationships, and environmental context. Maintaining an appropriate scope for the identification of new virulence factors can often be challenging. Especially since certain contexts, such as the introduction of at least one virulence factor in a population can significantly alter the landscape of the surrounding microbiome and the pathological potential of individual microbes, including those normally considered commensal. Crucially, few tools exist for probing microbial virulence in metagenomes, which constrains the development of culture-free methods in public health and epidemiology.\n\nWe found it difficult to manually identify and acquire datasets with sufficiently detailed metadata for this task. After identifying an appropriate metagenomic study, the data from both healthy and diseased human skin microbiomes was subjected to the HMM portion of the workflow (Figure 5). We found that assembling the sequencing reads was relatively fast but was still the limiting factor in the overall speed of the analysis. Although the workflow that we implemented seems logical, additional software improvements are necessary to improve tractability and obtain the desired results in a reasonable amount of time.\n\nA graphical representation of the workflow to predict the presence of virulence factors from metagenomic datasets.\n\nA depiction of the computational workflow used to identify antimicrobial resistance markers in metagenomic datasets.\n\nAMR has been identified as a global concern, as microorganisms worldwide gain the ability to resist drugs that have been used to treat common infections. Identifying AMR genes has been the focus of several organizations, leading to the curation of large bacteria-focused databases such as the Bacterial Antimicrobial Resistance Reference Gene Database, and a call for the characterization of these genes (Buckner et al. 2018). Plasmids, which are self-replicating elements, can contain AMR genes and as they are often transmissible through horizontal gene transfer, have become a concern for the global spread of antibiotic resistance (San Millan 2018). Given that a human microbiome sample includes thousands of species, surveillance and the detection of AMR genes is critical. Developing tools that can not only identify which AMR genes are present but that can also classify which bacterial species the AMR genes are likely from, and whether these AMR genes were likely derived from a plasmid, is extremely informative. The intersection of this information is of utmost importance as plasmids have been identified in the spread of AMR in specific bacterial families, such as Enterobacteriaceae and Enterococcaceae.\n\nWe designed each of the pipelines such that they generated a list of AMR gene “hits” from the sequencing reads used as input. In this case we used the publicly available human fecal gut SRA sample ERR1600439, available from NCBI. We observed that the number of hits varied depending on the algorithm that was used. Specifically, we found that magicBLAST identified 17 AMR candidates and MASH identified 45 AMR candidates. After guided assembly, each pipeline created a varied number of contigs, with magicblast creating seven, which was the most generated by any of the three algorithms.\n\nInfections caused by Enterococcus spp. present a major threat to public health as a leading cause of nosocomial infections. One major contributor to the severity of infections caused by enterococci is its frequent resistance to antimicrobial therapy. The association of resistance with mobile elements is of particular concern, as this can result in the rapid spread of AMR to previously susceptible isolates. Thus, the development of tools to identify mobile elements and their association with resistance genes will fill an important gap in current knowledge and provide a useful resource for public health and research applications.\n\nOur mobile element (“mobilome”) analysis was implemented as a computational workflow capable of identifying antimicrobial resistance genes that are flanked by active mobile elements (Figure 7). The tabular report that was generated by the workflow consisted of rows that contained information about each isolate-feature combination. The location, feature type, analysis method, name, and relevant BLAST statistics for each isolate-feature combination are reported in a tab-delimited text format. BLAST was used to compare each individual genome against each reference plasmid to determine if a particular reference plasmid is present in the sequence of a given isolate. The global coverage of each plasmid by each isolate is reported in a tab-delimited text format.\n\nMean RNA and DNA abundance values for Eschericia coli (left) and Bacteroides vulgatus (right). Each point represents a patient sample. Most patients have lower (relative) amounts of RNA from B. vulgatus compared to DNA; and the converse is true for E. coli.\n\nUse cases\n\nWe wanted to construct a pilot workflow within this cloud-based platform to rapidly and effectively preprocess and analyze metatranscriptomic data. This workflow could be used to support future analyses to distinguish between active and inactive/dormant bacterial taxa in a given sample. The aim for this use case was to determine whether we could detect such differences in bacterial populations using metatranscriptomics, and whether any biological insight could be derived from the results. While many interesting questions can be pursued by analyzing metatranscriptomic data, we decided to quantify differences between levels of DNA and RNA for a given gene--to distinguish between persistence and active metabolism.\n\nPersisters are often defined as metabolically inactive bacteria, characterized by arrested growth, low ATP levels, and low mRNA expression. Bacteria can enter a persistent state when exposed to supra-lethal concentrations of antimicrobial compounds, and can resume growth after the compound is removed from the environment (Fisher et al. 2017). Thus, persistence is a form of transient antibiotic tolerance, and can result in treatment failure and relapse of infection in the clinic (even in the absence of antibiotic resistance). It is therefore crucial to be able to detect persisters in clinical samples. One possible method of detection of persisters is genome-wide absence of RNA transcripts, but a presence of genomic DNA. This can be accomplished by considering the mean or median RNA vs DNA abundances across all pathways of each individual species (Figure 7).\n\nSimilar to how a human cell with one genome can differentiate into vastly diverse cell types, similar metagenomic compositions in microbial communities can have vastly different transcriptional/functional profiles (Franzosa et al. 2014). Many genes/pathways demonstrate a strong correlation between RNA and DNA abundance. However, there are also examples of pathways where DNA abundance does not vary much across samples, but RNA abundance does (Figure 8).\n\nArginine biosynthesis (left) and Fucose-rhamalose catabolism (right) pathways. Each point is a patient sample. For the arginine synthesis pathway, RNA and DNA abundance correlate well. However, for the fucose-rhamalose catabolism pathway is an example of low variability in terms of DNA abundance, but high variability in RNA abundance.\n\nRecent studies classified healthy vs diseased patients based on NGS data (Bang et al. 2019) (Pasolli et al. 2016). Early analysis of metatranscriptomic data has identified differentially-expressed pathways in periodontal disease when compared to healthy oral microbial communities (Jorth et al. 2014). Metatranscriptomic profiling data potentially has richer and more informative features than metagenomic or 16S data; since it provides information on which genes and pathways are being actively expressed, which may contribute to a host phenotype. We therefore selected the IBDMDB dataset for training ML models since it has metatranscriptomics data, and disease metadata.\n\nAs a pilot experiment, we trained seven machine learning models (decision tree, logistic regression, SVM, Naive Bayes, K-nearest neighbor, random forest and multi-layer perceptron) using the pathway abundance data extracted from the metatranscriptomics data from the HMP2 cohort in IBDMDB. A majority voting consensus model was also generated using the outputs of the 7 individual trained models (Figure 9). This workflow demonstrates the advantage of using metatranscriptomic data to stratify patients. We expect that this technology could be improved in the future to enable the accurate identification of persister/dormant bacteria that may contain virulence factors.\n\nThe training set (left) and test set (right). nonIBD: no Inflammatory Bowel Disease. UC: Ulcerative colitis. CD: Crohn's Disease.\n\n\nDiscussion\n\nThis work describes our efforts to demonstrate the usefulness of using existing cloud-based platforms to not only perform analytical tasks related to identifying markers of microbial virulence, but also to use these platforms to store the relevant data and metadata. Specifically, we generated proof-of-concept computational workflows for data storage and indexing, metabolic functions, virulence factors, antimicrobial resistance, and mobile element detection in enterococci.\n\nAs metadata and derived metadata become more descriptive and comprehensive in the future, we expect that data indexing and storage will continue to play an important role in data mining activities. The subtask that involved comparing the complexity of compressed sequence data and metadata provided a foundation upon which to build and improve. We anticipate that the degree of metadata compression will be reduced as the metadata becomes more expansive and less repetitive overall. The increased metadata complexity could approach the complexity of the sequencing data in the future. The increased amounts of metadata would therefore help with both identifying relevant datasets by applying filtering criteria as well as with predicting additional related datasets. Overall, efforts for continued data indexing, together with the association of relevant and informative metadata are imperative to facilitate the future use and re-use of public datasets. In addition, future meta-analyses that both increase sample size and augment knowledge will benefit from enhancing existing minimum metadata reporting standards.\n\nThe ability to accurately identify metabolic functions associated with the disease or non-disease state from metagenomic data will be useful as sequencing-based assays continue to gain traction in the clinic. When sufficient datasets and metadata are used to train the machine learning model, this proof of concept shows that the presence of a disease state, and potentially even the identification of the specific disease state, can be possible. Similarly, identifying gene patterns that are specific to particular disease states and their contribution to disease progression would be invaluable as we seek to better understand the underlying mechanism(s) of disease.\n\nWe envision the computational identification of potential virulence factors to be useful not only in current data mining applications, but also to identify potential microbial factors that could be impacting human health once sequencing becomes more routine in the clinical setting. The results from a similar workflow could be used to inform the best treatment strategy for a given individual.\n\nThe computational AMR gene detection workflow that we implemented can be useful in clinical contexts where time from sample collection to prescribed treatment is precious. For example, rather than waiting for minimum inhibitory concentration (MIC) assay results to be performed and reported by the clinical lab, sequencing a sample and running an AMR-detection pipeline could inform the clinician which AMR genes are present in the sample and could nicely complement the MIC results.\n\nThe mobilome analysis will be used in cases where users are interested in the transmissibility of genomic material, particularly related to antimicrobial resistance. Association of resistance genes with transposons, plasmids, or other mobile elements can yield valuable information on the transmission of resistance through bacterial populations. This can help track the emergence and spread of antimicrobial resistance on particular genetic elements.\n\nOverall, we found the NCBI hackathon for Microbial Virulence in the Cloud was extremely productive as measured by the various workflows, analyses, and results that were generated. These tools and results can be built upon by the scientific research community to improve existing methods and initiate important discussions about roadblocks and successes associated with our approach. For example, combining the various virulence detection methods could enable the rapid identification of emerging pathogens, while linking metabolism and signaling results with expression analyses could be used to identify novel indicator genes and therapeutic targets.\n\n\nConclusions and next steps\n\nWe are pleased with the publicly available workflows that were generated during this hackathon to build tools that can be applied to augment our understanding of microbial virulence and antimicrobial resistance. Potential next steps for this effort could include one or more of the following tasks: making the existing versions of pipelines and algorithms more robust, supporting the analysis of multiple input file types, applying and comparing the results from other algorithms, import workflow(s) into the NCBI Pathogen Detection Pipeline, dockerize tools and/or port them to external analysis platforms, improve data normalization methods, formalize the various database structure to facilitate data transfer across platforms, improve ease-of-use by developing web-based graphical user interfaces for a subset of tools, develop a database with the ability to take tab-delimited files containing metadata or other values and generate a table with standardized column names, and ensure the compatibility of tools, data structures, and containers across multiple cloud-based platforms.\n\nEach of the workflows implemented during this hackathon greatly benefited from the availability of public data in the cloud. The analyses that were performed incorporated statistical analysis to minimize bias and quantify observed variability. Beginning each analysis from data and algorithms hosted in the cloud shows the relevance of cloud-based resources such as Google Big Query for storing, retrieving, and mining large datasets and repositories. This approach and the underlying cloud platform are both scalable in a way that should allow the support of other organisms and additional metadata. Throughout this process the extreme importance of accurate, descriptive, and available metadata became apparent. In addition, we found that augmenting the current metadata for each experiment by identifying features such as the presence of AMR or other virulence factors could improve downstream reuse and analysis. In summary, this hackathon quickly generated a number of publicly available workflows that can contribute to future research efforts related to AMR, metagenomics, and metatranscriptomics.\n\n\nData availability\n\nThe data used for these projects were obtained from publicly accessible repositories, including NCBI, and are available in Table 1.\n\n\nSoftware availability\n\nPipelines and workflows available from:\n\n• Virulence AMR Data Index Resource: https://github.com/NCBI-Hackathons/Virulence_AMR_Data_Index_Resource/\n\n• Nasty Metagenomes: https://github.com/NCBI-Hackathons/Nasty_Metagenomes\n\n• MetaClaMP-ML: https://github.com/NCBI-Hackathons/MetaClaMP-ML\n\n• Virulence Factor Characterization: https://github.com/NCBI-Hackathons/Virulence_Factor_Characterization\n\n• Enterococcus Mobile Elements: https://github.com/NCBI-Hackathons/Enterococcus_Mobile_Elements\n\n• Enterococcus Mobile Elements: https://github.com/NCBI-Hackathons/Metatranscriptomics_Pilot\n\nArchived pipelines and workflows as at time of publication:\n\n• Virulence AMR Data Index Resource: https://doi.org/10.5281/zenodo.5227710 (DrCapt et al. 2021)\n\n• Nasty Metagenomes: https://doi.org/10.5281/zenodo.5361759 (Mendes et al. 2021)\n\n• MetaClaMP-ML: https://doi.org/10.5281/zenodo.5236567 (Elmassry et al. 2021)\n\n• Virulence Factor Characterization: https://doi.org/10.5281/zenodo.5338778 (Payne et al. 2021)\n\n• Enterococcus Mobile Elements: https://doi.org/10.5281/zenodo.5565736 (Ghtyson et al. 2021)\n\n• Metatranscriptomics Pilot: https://doi.org/10.5281/zenodo.5227692 (Defne & Busby 2021)\n\nLicense: MIT", "appendix": "Acknowledgments\n\nWe thank Gregory Tyson at the U.S. Food and Drug Administration, Center for Veterinary Medicine, Office of Research and Shurjo Sen at the Division of Genome Sciences, National Human Genome Research Institute. We also gratefully acknowledge NCBI and the U.S. National Institutes of Health for hosting this hackathon. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other federal agencies. These agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAltschul SF, Gish W, Miller W, et al.: Basic local alignment search tool. J. Mol. Biol. 1990; 215(3): 403–410. PubMed Abstract \| Publisher Full Text\n\nBang S, Yoo D, Kim S-J, et al.: Establishment and evaluation of prediction model for multiple disease classification based on gut microbial data. Sci. Rep. 2019; 9(1): 10189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuckner MMC, Ciusa ML, Piddock LJV: Strategies to combat antimicrobial resistance: anti-plasmid and plasmid curing. FEMS Microbiol. Rev. 2018; 42(6): 781–804. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "123011", "date": "03 Mar 2022", "name": "Rick Stevens", "expertise": [ "Reviewer Expertise Bioinformatics", "AI", "large-scale biological databases", "machine learning", "HPC" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report summarizes a hackathon that was held at NCBI in July of 2019. The participants in the group conducted pilot studies to explore building pipelines for identifying metabolic functions, virulence factors, AMR, mobile elements, and analyzing metatranscriptomic data. The workflows and software are listed in the article and are available for public use.\n\nOverall, while none of the workflows appear to be at a point of development where they stand alone as publishable entities, I thought that this was a fairly interesting article since it summarized the results of a seemingly productive hackathon. The paper is also a useful reference for various methods relating to these topics. In my opinion, there is a place for a publication of this type and that it should be accepted on its merit, assuming that it meets the requirements of this journal. My comments are fairly minor.\n\nI didn’t think the title fully matched what had been done in the hackathon.\n\nI found the justification of the need for the cloud to be tenuous, all of the other sections of the report and workflows have some biological justification, and could in theory stand alone. If that part is to be kept, it seems like it should go last with an explanation of how to use the data in the cloud (assuming that was built).\n\nWe know what AMR stands for, but please define “AMRs”. Is this genes, proteins, etc? “AMRs” also appears at the bottom of figure 6.\n\nIn the mobile elements section, “Figure 7” has the wrong figure number.\n\nFigure 9 would be easier to read with the raw counts instead of scientific.\n\nAccuracy or F1 scores of the transcriptomic disease model(s) and other models could be stated in the results\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "243832", "date": "22 May 2024", "name": "Kevin Tyler", "expertise": [ "Reviewer Expertise Editor of Virulence" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this was probably a useful piece of work for those involved and may have progressed the field. It's delayed indexing may still be helpful to some but has clearly diminished its value. I am sure the hackathon itself is a useful forum and would encourage its participants to run another in this area in future years but I would advise that any publication from it should be rapid to provide maximum value. We launch a blog at Virulence this year (PathogenCity) and would welcome a blog - or even potentially an editorial to announce any new hackathon in this area in due course. Unfortunately, the workshop reported here was quite a while ago now (August 2019) and a lot of work since then has largely superceded these analyses and tools so readers will have to read forward further to catch up with the status quo. Actually, it would be interesting to know how many of the high profile papers in this area - many of which in process or have come out over the last couple years are rooted in this workshop and tools associated with it as all the references cited are pre-workshop. I'm not sure to what extent the methods being used are drawn from events such of these or are from parallel developments at multiple research centres...Mostly the report reads well - I note the term AMR genes, is widely abbreviated as ARGs these days.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-160
https://f1000research.com/articles/10-525/v1	01 Jul 21	{ "type": "Research Article", "title": "Impact of freeze-thaw cytoablation on aqueous outflow patterns in ex vivo anterior chamber perfusion cultures and whole eyes", "authors": [ "Raoul Verma-Fuehring", "Mohamad Dakroub", "Alicja Strzalkowska", "Piotr Strzalkowski", "Hong Han", "Jost Hillenkamp", "Nils A. Loewen", "Raoul Verma-Fuehring", "Mohamad Dakroub", "Alicja Strzalkowska", "Piotr Strzalkowski", "Hong Han", "Jost Hillenkamp" ], "abstract": "Background: Porcine eyes have been widely used as ex vivo models in glaucoma research, as they share similar features with human eyes. Freeze-thawing is a non-invasive technique that has been used to obliterate living cells in anterior segment ex vivo cultures, to prepare them for further research such as cellular repopulation. This technique has previously been shown to reduce the intraocular pressure (IOP) in porcine eyes. The aim of this study was to investigate whether freeze-thaw cytoablation causes corresponding canalogram outflow changes in perfused anterior segment cultures (AFT) and whole porcine eyes (WFT). We hypothesized that the known IOP drop in AFT after trabecular meshwork ablation by freeze-thaw would be accompanied by a similarly large change in the distal outflow pattern. Methods: Two-dye (fluorescein and Texas red) reperfusion canalograms were used to compare the outflow time before and after two -80°C cycles of freeze-thaw. We assigned 28 freshly enucleated porcine eyes to four groups: perfused anterior segment dye controls (ACO, n = 6), perfused whole eye dye controls (WCO, n = 6), freeze-thaw treated anterior segment cultures (AFT, n = 10), and freeze-thaw treated whole eyes (WFT, n = 6). Results: In control groups ACO and WCO, the two different dyes had similar filling times. In AFT, the outflow pattern and filling times were unchanged. In WFT, the temporal superior quadrant filled more slowly (p = 0.042) while all others remained unchanged. The qualitative appearance of distal outflow spaces was altered only in some eyes. Conclusions: Freeze-thaw cytoablation caused neither loss nor leakage of distal outflow structures. Surprisingly, the loss of an intact trabecular meshwork over the entire circumference did not result in a general acceleration of quadrant outflow times. The results validate freeze-thawing as a method to generate an extracellular matrix without major structural changes.", "keywords": [ "freeze-thaw", "porcine eyes", "trabecular meshwork", "canalography" ], "content": "Introduction\n\nThe trabecular meshwork represents a location of great interest for glaucoma research, and non-invasive methods are needed to decellularize it and convert it into a scaffold for cellular transplantation. As porcine eyes share several important features with human eyes1–3, can be used to mimic surgical interventions4,5 and different glaucoma types, including secondary open angle6 and angle closure glaucomas7, they have been widely used as ex vivo models in glaucoma research. In contrast to human donor eyes, they are also ubiquitously available and remain responsive to stimuli and drugs when freshly harvested. This allows the exploration of anatomical functions3,8 and the effects of biologicals9, drugs10, and surgical interventions11. Corresponding outflow changes can be studied with high spatial and temporal resolution5,12,13.\n\nAb interno trabeculectomy of the nasal circumference increases outflow in whole porcine eyes as well as anterior segment cultures5. Outflow is changed mostly at the site of ablation but also enhanced circumferentially5. In contrast to ab interno trabeculectomy, freeze-thaw decellularization is a nonspecific and site-agnostic method to remove all living cells from eye cultures to ready them for the transplantation and study of cells of interest14,15. In perfused porcine anterior segments, this is accompanied by a significant reduction in intraocular pressure (IOP)16. Freeze-thawed scaffolds may be easier to generate than artificial three-dimensional trabecular meshwork structures17–19.\n\nHere, we hypothesized that subjecting porcine eyes to freeze-thaw decellularization would result in faster outflow throughout the entire circumference and alter the outflow pattern due to a loss of endothelial integrity of the distal outflow tract.\n\n\nMethods\n\nTwenty-eight porcine eyes were assigned to one of four groups: anterior segment control (ACO, n = 6), and whole eyes control (WCO, n = 6), freeze-thaw anterior segment (AFT, n = 10), freeze-thaw whole eyes (WFT, n = 6). Eyes in the experimental group (AFT and WFT) underwent two cycles of freeze-thaw at -80°C. Reperfusion canalograms were carried out in the two control groups that did not undergo freeze-thaw (ACO and WCO), as well as before and after freeze-thawing in the experimental groups (AFT and WFT). No live vertebrate animals were used in this study. Pig eyes were obtained from a local abattoir. As a result, no ethics approval was required.\n\nFreshly enucleated porcine eyes were obtained from a local abattoir (Landschlachterei Issing, Retzbach, Bavaria, Germany) and processed within three hours of death. All eyes were placed in a 5% povidone-iodine solution for three minutes, and rinsed twice with phosphate-buffered saline (PBS) (Dulbecco’s Phosphate Buffered Saline, Sigma-Aldrich, St. Louis, Missouri, USA). The laterality (left versus right eyes) was determined by examining the extraocular muscles and the shape of the cornea. The extraocular tissues were removed for all eyes. AFT and ACO eyes were then bisected at the equator. This was followed by the removal of the vitreous body, lens, and uvea. The eyes were then rinsed with PBS, mounted on perfusion dishes and infused with Dulbecco’s Modified Eagle Medium (DMEM) (Gibco/Life Technologies, Carlsbad, California, USA) at a constant pressure of 15 mmHg using a 20 ml syringe at a height of 20 cm above the perfusion chamber.\n\nWhole eyes were mounted facing up using a specimen vial (Wheaton CryoElite Tissue Vial #W985100, Wheaton Science Products, New Jersey, USA). A 30G needle was subsequently inserted bevel-up into the anterior chamber through the temporal cornea. Perfusion was maintained for 20 minutes in both anterior segments and whole eyes.\n\nPerfusion with fluorescein: After 10 minutes of perfusion with DMEM, an anterior chamber exchange was performed in AFT and ACO by opening the outflow and allowing the anterior chamber to empty before the infusion was switched to 0.017 mg/ml fluorescein (fluorescein solution 10%, Alcon, Freiburg, Switzerland). The outflow port was then closed and the canalogram images were captured at 30-second intervals. In whole eyes, a safe anterior chamber exchange could not be performed without injuring the lens capsule and was therefore omitted.\n\nFreeze-thaw cycles: The treatment groups AFT and WFT underwent two cycles of freeze-thaw. These consisted of freezing at - 80°C for two hours, followed by thawing at room temperature for one hour16.\n\nPerfusion with Texas red: Another full anterior chamber fluid exchange was performed in anterior segments after the freeze-thaw cycles by connecting a 20 ml reservoir containing 0.28 mg/ml Texas red (Sulforhodamine 101, 25 mg crystalline solid, Hycultec, Beutelsbach, Germany) to the anterior segments. Fluorescent images were acquired. In whole eyes, the same protocol was used but without an anterior chamber exchange.\n\nFluorescent images were captured using a stereomicroscope (Olympus SZX, Olympus K.K., Tokyo, Japan) equipped with a CoolLED pE-300 (CoolLED Limited, London, UK) white illumination unit and an 0.5x objective lens. Canalograms were processed with the Olympus cellSens Dimension 2.3 software (Olympus K.K., Tokyo, Japan). Images were recorded in a time-lapse every 30 seconds for 20 minutes with a resolution of 680 x 510 pixels in 8-bit grayscale. Exposure time was set to 57.14 ms for fluorescein and 344.80 ms for Texas red.\n\nThe canalograms were analyzed for filling times of each quadrant: nasal-superior (NS), nasal-inferior (NI), temporal-superior (TS) and temporal-inferior (TI). Filling time was defined as the time elapsed between the first visualization of the dye in the episcleral veins and their complete filling.\n\nSagittal specimen sections were obtained and fixed with 4% paraformaldehyde in PBS for 24 hours. After rinsing them three times in PBS, they were embedded in paraffin, sectioned at 6-micron thickness, and stained with hematoxylin and eosin.\n\nData was analyzed using SPSS Statistics (Version 26, IBM, New York, USA). Filling times were reported in seconds. We calculated means and standard deviations. A Shapiro-Wilk test was deployed to check for a normal distribution of acquired filling times. We used a paired, one-tailed t-test and Wilcoxon signed-rank test to compare the mean values of the filling times before and after freeze-thaw. The one-way analysis of variance (ANOVA) was run to compare filling times between quadrants. For all our statistical analyses, a p-value of 0.05 or less was considered statistically significant. A post-hoc power analysis was carried out using GPower (Version 3.1.9.7., Heinrich Heine University, Düsseldorf, Germany).\n\n\nResults\n\nA total of 28 eyes were included in the analysis consisting of 16 right eyes and 12 left eyes. There were six eyes in each of ACO, WCO, and WFT, and ten eyes in AFT. Within the control groups, ACO and WCO, there were no significant differences between the quadrant filling times (all p > 0.05)20. Additionally, both dyes showed similar filling times within each quadrant (Table 1, all p > 0.05).\n\nTS = temporal superior, TI = temporal inferior, NS = nasal superior, NI = nasal inferior, ACO = perfused anterior segment dye controls, WCO = perfused whole eye dye controls.\n\nFigure 1 shows the filling times of anterior segments pre- and post-freeze thaw (AFT). In these eyes, quadrant TS tended to have a faster average filling time than the other three quadrants, both pre (270 ± 136 s) and post freeze-thaw (441 ± 371 s), but without reaching statistical significance (p > 0.05). Moreover, freeze-thaw did not cause a significant change in the filling times of any quadrant (p > 0.05).\n\nTS = temporal-superior, TI = temporal-inferior, NS = nasal-superior, NI = nasal-inferior.\n\nFigure 2 depicts the filling times before and after freeze-thaw in WFT eyes. There was no significant difference in the filling times between quadrants (p = 0.401). TS showed the shortest filling time before freeze thaw with an average of 162 ± 101 s. The filling time of this quadrant significantly increased after freeze-thaw (355 ± 175 s, p = 0.002). None of the other WFT quadrants showed a significant change in filling times after freeze-thawing.\n\nComparison of filling times before and after freeze-thaw in whole eyes TS = temporal-superior, TI = temporal-inferior, NS = nasal-superior, NI = nasal-inferior, = significant.\n\nFigure 3 depicts the canalogram of an AFT eye. In this eye, the post-freeze-thaw canalogram demonstrated additional temporal outflow channels that were not visible before (TI) or only partially filling (TS).\n\nFreeze-thaw treated anterior segment cultures (AFT) canalogram before (A) and after (B) freeze-thawing. Example of perfused anterior segment culture, in which outflow channels that were not functional become visible after freeze thaw (white arrows). TS = temporal-superior, TI = temporal-inferior, NS = nasal-superior, NI = nasal-inferior.\n\nFigure 4 shows canalograms of WFT eye pre- and post- freeze-thaw. Visually, both images show a similar filling pattern after 20 minutes of perfusion. In this case, fluorescence was captured mainly in the nasal episcleral veins.\n\nFreeze-thaw treated whole eyes (WFT) canalogram before (A) and after (B) freeze-thawing. After a 20-minute infusion with fluorescein, a fluorescent signal was detected in the nasal-superior (NS) quadrant (A). This outflow pattern experienced only minor changes (white arrows). The shadow of the infusion needle (N) is visible on the right (temporal) side. TS = temporal-superior, TI = temporal-inferior, NS = nasal-superior, NI = nasal-inferior.\n\nHistological analyses demonstrated unchanged trabecular beams but without intact trabecular meshwork cells (Figure 5). Compared to control eyes, there were no noticeable changes to the tissue surrounding the angular aqueous plexus. The lumina of this plexus, including larger, Schlemm’s canal like-segments remained intact.\n\nSagittal histology sections comparing the angular aqueous plexus in control (A) and freeze-thaw ablated eyes (B). The TM beams and SCLS remain intact after freeze-thaw. TM = trabecular meshwork, SCLS = Schlemm’s canal-like segments (red arrows).\n\nThe post-hoc power analysis of the filling times pre- and post- freeze-thaw for each group is presented in Table 2. In general, results revealed a low power for most of the quadrants. Within the experimental groups, the TS quadrant comparison had the highest power with values of 0.5 and 0.75 in AFT and WFT, respectively. All other quadrants in both groups had a power below 0.3.\n\nTS = temporal-superior, TI = temporal-inferior, NS = nasal-superior, NI = nasal-inferior, ACO = perfused anterior segment dye controls, WCO = perfused whole eye dye controls, AFT = freeze-thaw treated anterior segment cultures, WFT = freeze-thaw treated whole eyes\n\n\nDiscussion\n\nWe have developed a freeze-thaw protocol to decellularize anterior segments and to obtain a three-dimensional matrix to transplant modified trabecular meshwork (TM) cells or other cells under study16,21. This approach might generate ex vivo models for genetically altered, glaucomatous TM cells or primary cells harvested in excisional ab interno trabeculectomy21–23. Surprisingly, freeze-thawing did not alter the vascular drainage spaces qualitatively in a significant way. Rather, dyes remained mostly intravascularly within the time observed without any noticeable differences on average as detectable with these methods. Fluorescein has a molecular size of 376 Da and sulforhodamine 101 acid chloride a size of 625 Da, small enough to pass through the TM relatively quickly, yet large enough to be confined to the intravascular space for minutes24–26. Histologic analyses also revealed a conserved TM structure16,21. This preserved scaffold along with the unchanged outflow vessels suggests freeze-thawing as a technique to produce realistic seeding matrices for trabecular meshwork transplantation.\n\nBecause freeze-thaw had previously been shown to cause an IOP drop of about 30% in porcine eyes16, we expected to see faster outflow throughout the 360 degrees of angular aqueous plexus of both anterior segments and whole eyes. Surprisingly, this was also not the case. The only quadrant in which there was a statistically significant difference was the temporal superior quadrant of whole eyes, and we observed an increase rather than a decrease in filling time. It is possible that freeze-thawing causes relatively small changes per trabecular meshwork area and that our study missed those due to the large standard deviation. However, these small changes might have a large cumulative impact and lower IOP16 because they occur over the entire circumference. For instance, flow in TI and NS had a lower average flow time after freeze-thaw compared to before, but without reaching statistical significance. Moreover, due to their moderate molecular size, movement of fluorescein and sulforhodamine through an intact trabecular meshwork might not be fundamentally different from movement through remnants of disrupted trabecular meshwork cells or their debris27. Additionally, our methods might miss diffuse fluid movements directly through the sclera that is akin to uveoscleral outflow.\n\nWe observed a longer post freeze-thaw filling time in the temporal superior quadrant of whole eyes. It is likely that intraocular tissues that are present in whole eyes, but are missing in anterior segments28, were disrupted by the freeze-thawing cycles. Their debris might have caused the delayed filling27, in particular iris pigment6,29,30. Pigment can lead to a slow increase of IOP over hours to days6 while larger debris from cell death can have a more immediate effect on the trabecular meshwork27.\n\nOne limitation is that we did not measure IOP because a goal of this study was to assess freeze-thawing in anterior segment cultures in comparison to whole eyes; however, the latter cannot easily be connected to a pressure transducer. Another limitation is that the eyes were not incubated for three days as we have done previously13. Due to the compression mount, outflow in anterior segments perfusion cultures is initially impaired, but this effect vanishes within less than three days of culture16. Finally, the 28 eyes used in this study were only able to provide a moderately low testing power to detect an outflow difference, providing further evidence that any flow difference caused must not be large.\n\nIn conclusion, we found no major outflow differences caused by freeze-thaw treatment of anterior segments or whole eyes. These results validated freeze-thawing as a method to generate a three-dimensional seeding matrix without losing distal outflow tract vessels.\n\n\nData availability\n\nFigshare: RawData_FreezeThaw.csv, https://doi.org/10.6084/m9.figshare.14610579.v120\n\nThe project contains the following underlying data:\n\n- RawData_FreezeThaw.csv (raw data of freeze-thaw in porcine eyes. filling times assessed using fluorescent canalography before and after freeze thaw)\n\nFigshare: Original Images FT, https://doi.org/10.6084/m9.figshare.14769888.v131.\n\n- Original, unedited image files underlying our results and figures\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nSanchez I, Martin R, Ussa F, et al.: The parameters of the porcine eyeball. Graefes Arch Clin Exp Ophthalmol. 2011; 249(4): 475–82. PubMed Abstract \| Publisher Full Text\n\nMcMenamin PG, Steptoe RJ: Normal anatomy of the aqueous humour outflow system in the domestic pig eye. J Anat. 1991; 178: 65–77. PubMed Abstract \| Free Full Text\n\nSaraswathy S, Tan JCH, Yu F, et al.: Aqueous Angiography: Real-Time and Physiologic Aqueous Humor Outflow Imaging. PLoS One. 2016; 11(1): e0147176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParikh HA, Loewen RT, Roy P, et al.: Differential Canalograms Detect Outflow Changes from Trabecular Micro-Bypass Stents and Ab Interno Trabeculectomy. Sci Rep. 2016; 6: 34705. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoewen RT, Brown EN, Scott G, et al.: Quantification of Focal Outflow Enhancement Using Differential Canalograms. Invest Ophthalmol Vis Sci. 2016; 57(6): 2831–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDang Y, Waxman S, Wang C, et al.: A porcine ex vivo model of pigmentary glaucoma. Sci Rep. 2018; 8(1): 5468. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHong Y, Wang C, Loewen R, et al.: Outflow facility and extent of angle closure in a porcine model. Graefes Arch Clin Exp Ophthalmol. 2019; 257(6): 1239–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkiyama G, Saraswathy S, Bogarin T, et al.: Functional, structural, and molecular identification of lymphatic outflow from subconjunctival blebs. Exp Eye Res. 2020; 196: 108049. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBirke MT, Birke K, Lütjen-Drecoll E, et al.: Cytokine-dependent ELAM-1 induction and concomitant intraocular pressure regulation in porcine anterior eye perfusion culture. Invest Ophthalmol Vis Sci. 2011; 52(1): 468–75. PubMed Abstract \| Publisher Full Text\n\nWaxman S, Wang C, Dang Y, et al.: Structure-Function Changes of the Porcine Distal Outflow Tract in Response to Nitric Oxide. Invest Ophthalmol Vis Sci. 2018; 59(12): 4886–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDang Y, Wang C, Shah P, et al.: Outflow enhancement by three different ab interno trabeculectomy procedures in a porcine anterior segment model. Graefes Arch Clin Exp Ophthalmol. 2018; 256(7): 1305–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaxman S, Loewen RT, Dang Y, et al.: High-Resolution, Three-Dimensional Reconstruction of the Outflow Tract Demonstrates Segmental Differences in Cleared Eyes. Invest Ophthalmol Vis Sci. 2018; 59(6): 2371–2380. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoewen RT, Brown EN, Roy P, et al.: Regionally Discrete Aqueous Humor Outflow Quantification Using Fluorescein Canalograms. PLoS One. 2016; 11(3): e0151754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurk J, Erbe I, Berner D, et al.: Freeze-thaw cycles enhance decellularization of large tendons. Tissue Eng Part C Methods. 2014; 20(4): 276–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu X, Zhao G, Shu Z, et al.: Quantification of Intracellular Ice Formation and Recrystallization During Freeze-Thaw Cycles and Their Relationship with the Viability of Pig Iliac Endothelium Cells. Biopreserv Biobank. 2016; 14(6): 511–9. PubMed Abstract \| Publisher Full Text\n\nDang Y, Waxman S, Wang C, et al.: Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model. PeerJ. 2017; 5: e3629. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorrejon KY, Papke EL, Halman JR, et al.: Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model. Biotechnol Bioeng. 2016; 113(6): 1357–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorrejon KY, Pu D, Bergkvist M, et al.: Recreating a human trabecular meshwork outflow system on microfabricated porous structures. Biotechnol Bioeng. 2013; 110(12): 3205–18. PubMed Abstract \| Publisher Full Text\n\nTorrejon KY, Papke EL, Halman JR, et al.: TGFβ 2-induced outflow alterations in a bioengineered trabecular meshwork are offset by a rho-associated kinase inhibitor. Sci Rep. 2016; 6: 38319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVerma-Fuehring R: RawData_FreezeThaw.csv. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.14610579.v1\n\nWaxman S, Wang C, Dang Y, et al.: Tissue-Engineered Anterior Segment Eye Cultures Maintain Intraocular Pressure. Medicine & Pharmacology. 2020. Publisher Full Text\n\nRamjiani V, Mudhar HS, Julian T, et al.: Sampling trabecular meshwork using TrabEx. BMC Ophthalmol. 2021; 21(1): 138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwaminathan SS, Monsalve P, Zhou XY, et al.: Histologic Analysis of Trabecular Meshwork Obtained from Kahook Dual Blade Goniotomy. Am J Ophthalmol. 2018; 192: 198–205. PubMed Abstract \| Publisher Full Text\n\nZhao X, Belykh E, Cavallo C, et al.: Application of Fluorescein Fluorescence in Vascular Neurosurgery. Front Surg. 2019; 6: 52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh JK, Dhawahir FE, Hamid AFA, et al.: The use of dye in ophthalmology. J Audiov Media Med. 2004; 27(2): 62–7. PubMed Abstract \| Publisher Full Text\n\nPark SA, Jeong S, Choe YH, et al.: Sensing of Vascular Permeability in Inflamed Vessel of Live Animal. J Anal Methods Chem. 2018; 2018: 5797152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBahler CK, Fautsch MP, Hann CR, et al.: Factors influencing intraocular pressure in cultured human anterior segments. Invest Ophthalmol Vis Sci. 2004; 45(9): 3137–43. PubMed Abstract \| Publisher Full Text\n\nJohnson DH, Tschumper RC: Human trabecular meshwork organ culture. A new method. Invest Ophthalmol Vis Sci. 1987; 28(6): 945–53. PubMed Abstract\n\nWang C, Dang Y, Shah P, et al.: Intraocular pressure reduction in a pigmentary glaucoma model by Goniotome Ab interno trabeculectomy. PLoS One. 2020; 15(4): e0231360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDang Y, Waxman S, Wang C, et al.: Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma [version 2; peer review: 2 approved]. F1000Res. 2018; 7: 174. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVerma-Fuehring R: Original Images FT. figshare. Figure. 2021. http://www.doi.org/10.6084/m9.figshare.14769888.v1" }	[ { "id": "89485", "date": "23 Aug 2021", "name": "Christian van Oterendorp", "expertise": [ "Reviewer Expertise glaucoma", "aqueous drainage" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 study on the influence of freeze-thaw decellularisation on the aqueous outflow tract in porcine eyes. They hypothesised that freeze-thaw would result in a higher outflow rate and changes to outflow pattern.\nThe study was designed as a longitudinal study, which was achieved by using two different fluorophores to conduct the experiments pre- and post-freeze-thaw in the same eyes. Filling time was used as surrogate parameter for the volumetric flow rate. This was supplemented by qualitative histological studies of the angular aqueous plexus.\nThe experimental setup is sound and the data is presented in a well structured fashion. To increase the transparency of the data the authors may contemplate choosing scatter plots with connecting lines for each pair of pre-post-data instead of bar and whisker plots. Thus, the reader would be able to see the change of filling time for each individual eye.\nWhile the main focus of data analysis was on the filling time, the authors show only 2 examples of changes of the outflow pattern (fig. 3+4). Quantifying these changes systematically would be desirable, because figure 3 suggests that the total cross sectional area of the aqueous drainage system has increased. Thus, the total volumetric flow rate of the eye (at constant perfusion pressure) could have changed despite a constant filling time of each individual vessel. The observation presented in figure 3 should also be further discussed regarding possible mechanisms of opening previously closed outflow channels.\nOne last point in regards the experimental setup: The authors used a 30G needle to perfuse the anterior chamber. Depending on the outflow rate of the eyes, the small internal diameter of the needle may render the needle an element of significant flow resistance, which in turn may result in an IOP lower than the hydrostatic pressure applied with the 20ml syringe at 20 cm height.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "91189", "date": "22 Sep 2021", "name": "VijayKrishna Raghunathan", "expertise": [ "Reviewer Expertise Trabecular meshwork mechanobiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 by Verma-Fuehring et al documenting the impact that freeze-thawing has on outflow time across the TM in porcine eyes. The study is largely conducted in a simple and effective manner, but could perhaps be improved with some clarifications.\nThe authors utilized a slow method of freezing anterior segment and whole globes through freezing in -80C. Due to variations in tissue thickness and permeability differences, perhaps a quicker freezing method could have been utilized to minimize such artifacts. e.g. rapid freezing using Liquid N2. Can the authors please clarify the choice of method used?\n\nAfter freeze-thaw, the authors choose to demonstrate through H&E staining and canalogram that fluid filling rates were unchanged. Subsequently they infer that this implies no structural changes were observed in the TM and hence the model may be adequate. However, no evidence to demonstrate changes in ECM composition (if any, or lack thereof) and organization is provided. This is particularly important since free-thaw techniques generally influence GAGs and other secretory proteins in tissues , at least as reported in non-ocular literature. This become even more important considering ECM composition and GAGs help maintain cellular phenotype and can influence differentiation. Can the authors please comment.\n\nFreeze-thaw indeed can cause cell lysis and loss of cells, as evident in their H&E staining. However, can the authors comment how one might go about removing debris and washing off porcine immunogenic/antigens that when human cells may be transplanted (as indicated for intended application) may not react to?\n\nCan the authors comment if they have attempted a more localized free-thaw technique (e.g. a cryo probe) that may not affect the integrity of the entire tissue and if this may be more favorable for such a model?\n\nShould the authors have the images, the manuscript may benefit in inclusion of a kymograph-like image demonstrating the appearance of the fluorescein dye to show fill timings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-525
https://f1000research.com/articles/11-158/v1	08 Feb 22	{ "type": "Opinion Article", "title": "All eyes on COVID-19, let's not forget Tuberculosis", "authors": [ "Vassia Schiza", "Yupei Xiao", "Andrea Tattersall", "Vassia Schiza", "Yupei Xiao" ], "abstract": "Tuberculosis (TB) is a severe global threat killing more than one million people annually (WHO). With a successful TB control programme in place, there has been a decrease in the number of TB cases and deaths globally over recent years. The World Health Organisation (WHO) End TB Strategy has been momentously shocked by the COVID-19 pandemic and it seems that any success made over recent years is likely to be reversed. We are now more than one year into the pandemic, and the effect COVID-19 has had on TB services is devastating. Hospitals typically dedicated to TB have been converted to COVID-19 hospitals and diagnostic laboratories focus on COVID-19 testing rather than TB. Delivery of TB care is being prioritised for people who have active TB disease whereas prevention and diagnosis of latent TB infection (LTBI) is being put on hold. This pause can lead to an increase in TB cases and transmission. Here, we discuss the connection between SARS-CoV-2 infection and latent TB and highlight the importance of TB prevention management in LTBI post-COVID-19 patients. Community engagement and contact tracing are of high importance in fighting TB in the post-COVID19 era. Getting back on track with TB progress is essential, thus further modelling on the COVID-19 impact on TB burden and its determinants is critical.", "keywords": [ "covid-19", "tuberculosis", "latent TB infection", "prevention", "treatment", "community engagement", "contact tracing" ], "content": "Introduction\n\nAs the COVID-19 pandemic continues to unfold, all eyes are on diagnostics, therapeutics, and vaccination programs to fight the virus and reduce cases, hospitalisations, and deaths. Heavy focus on a single pathogen has reshaped activities across the diagnostic landscape with basic health services and screening programmes being disrupted. What effect has this disruption had on a much older infectious and endemic disease – Tuberculosis (TB)? The focus on the COVID-19 pandemic has had a direct impact on the TB care cascade, with considerable delays in diagnosis and interruption to treatment, as well as a decrease in demand and access to treatment.1 Mathematical modelling conducted by multiple research groups estimates the impact of the COVID-19 pandemic on TB incidence and mortality, suggesting an increase of around 5-15% over the next 5 years.1–5 TB remains a global health emergency affecting mostly the poorest and most vulnerable, and we believe that now more than ever is the time to focus global efforts on the growing TB epidemic.\n\nTB is caused by the bacillus Mycobacterium tuberculosis, which is spread when people who are infected expel bacteria into the air; for example, by coughing.6 TB infection exists in two phases: latent TB infection (LTBI) when people are asymptomatic and cannot pass it on to other people, and an active phase when they develop symptoms and become ill.6 Active TB can affect the lungs (pulmonary TB) with symptoms such as coughing blood or sputum, pain in the chest and fever; but it can also spread to other sites (extrapulmonary TB), in which case symptoms would be localised in those areas.6 TB remains the number one cause of death from a single infectious agent globally – killing approximately 4,000 people per day, and 1.4 million annually.6 This is a massive human and societal toll for a curable and preventable disease. Of the 10 million global cases of TB, South-East Asia has around 44%, Western Pacific 18%, and Africa around 25%, while America has 2.9% and Europe has the lowest number of cases at 2.5%.6 In the UK, there is a specific TB control programme in place, with tremendous TB control efforts which has led to a decline in the number of TB cases to approximately 4,500 new cases annually.7 However, according to Public Health England (PHE), the UK has the second highest mortality from TB in Western Europe, which is five times higher than in the US.\n\n\nCOVID-19 impact on TB services\n\nOne year on from the start of the pandemic, we are now seeing the catastrophic effect that COVID-19 has had on the global TB burden and TB services. Policies adopted in response to the pandemic have disrupted routine TB medical practices and converted designated TB wards to COVID-19 wards, and many respiratory specialists efforts have been diverted away from TB.8 In the UK, TB services have had to prioritise and focus on delivering care for TB disease rather than prevention (such as the management of latent, non-transmissible TB infection),9 and diagnostic laboratories are prioritising COVID-19 testing instead of TB testing. As a result, there has already been a drop in TB notifications, with a diagnostic delay for suspected TB patients; the substantial delays in TB diagnosis may increase community transmission of TB and development of drug-resistant TB. A decrease has also been observed in the number of TB patients seeking help due to fear of SARS-CoV-2 transmission in health facilities. Additionally, in paediatric TB, which progresses silently and needs to be reviewed regularly, routine clinics have been cancelled, and paediatric care beds moved to COVID-19 wards.9 Moreover, the reallocation of resources towards COVID-19 may lead to delays to innovative TB trials, which could halt the progress made towards new TB drugs, diagnostics, and vaccines.\n\n\nAre we meeting the ambitious targets of the WHO End TB Strategy?\n\nAs part of the efforts to end the global TB epidemic, the WHO has set the END TB strategy to reduce TB incidence rate and deaths as well as catastrophic costs by 2035, with interim milestones set for 2020 and 2025. The strategy puts patients at the heart of service delivery, with strong participation from government, communities, and private stakeholders, and focuses on research and innovation.10 Prior to the COVID-19 pandemic many high TB burden countries were not on track to reach the 2020 milestones of the End TB Strategy. There was, however, clear progress in reducing the TB incidence rate as well as the number of deaths globally as shown in both the Global TB Report 20206 and United Nations (UN) Political Declaration11 on TB. COVID-19, however, is likely to cause a dramatic setback and reverse this progress. According to a modelling analysis commissioned by the Stop TB Partnership, a three-month lockdown and a ten-month restoration period could lead to an additional 6.3 million TB cases and 1.4 million TB deaths globally between 2020 and 2025.12 There was a 25-50% drop in TB case detection over three months in several high TB burden countries including India and Indonesia in 2020. This would result in 200-400,000 additional TB deaths in 2020, which is equivalent to the annual mortality for 2012.6 One of the commitments of the global leaders at the UN high-level meeting on TB in 2018 was to ensure access to preventive TB treatment for 24 million contacts of active TB patients by 2022.11 It seems that the COVID-19 pandemic could prove a major setback for such efforts. In 2018-2019 only 1 million people were treated for TB, which is only 35% of the 40 million target by 2022 and just over one-fifth of the five year target started TB preventive treatment.11 These setbacks on TB control jeopardise our ability to meet the goals set by the End TB Strategy and getting the world back on track for TB control needs our attention more than ever in both adults and children.\n\n\nCursed duet\n\nBoth TB and COVID-19 are respiratory pathogens that are spread through the air and primarily attack the lungs with symptoms of cough, fever and difficulty breathing. The key question is whether coinfection with both TB and COVID-19 is more severe than individual infection with either one. A recent meta analysis suggested that the risk of death in COVID-19 and TB coinfected patients is 2.17 times higher and the risk of recovery is 25% lower than in those without TB.13 Thus, coinfection with TB has shown poorer treatment outcomes and increased morbidity and mortality in COVID-19 patients.13 COVID-19 has also been shown to contribute to the deterioration of TB patients by worsening symptoms, in some cases leading to death.14 Another study, however, investigated 20 patients co-infected with TB and COVID-19 in Italy, and showed that active TB appears to be clinically manageable with only one death in the study.15 In immunocompromised patients with underlying comorbidities, COVID-19 leads to severe pneumonitis and long-term lung damage. One should also keep in mind that TB patients often have underlying comorbidities such as immunocompromised conditions like HIV/AIDS, diabetes, and lung damage. Whilst diabetics have a high risk of developing latent TB, diabetes has now emerged as a major co-morbidity for COVID-19 as well.16 It is of extreme importance to consider that such comorbidities could act as risk factors for developing severe COVID-19 or active TB, or both. Patients with active TB, or those who have previously recovered from it, are left with fibrotic, scarred lungs and compromised lung function. As such, the post-COVID-19 effects can be devastating in this patient population. Adversely, in most COVID-19 autopsy series published, diffuse alveolar damage (DAD) is the most common finding. “This post-COVID pulmonary fibrosis, superimposed on the fibrosis caused by the sequelae of pulmonary TB, is likely to result in even more devastating disability”.17 What is tremendously important now is to have a plan for managing patients with respiratory symptoms suspected for both TB and COVID-19.\n\n\nPost COVID-19 patients: From LTBI to active TB\n\nA number of people with LTBI get ill when their immune system becomes weak for another reason. There is a worrisome connection between SARS-CoV-2 infection and latent TB. Clinical studies have shown that co-infection with SARS-CoV-2 accelerates TB progression by weakening host immunity.18,19 Inflammation is the major driving pathology for severe disease in COVID-19 patients, and anti-inflammatory drugs such as corticosteroids (CST) are used to treat severe COVID-19 cases.20 However, CSTs work by suppressing the immune system, and as such treatment with CST poses a significant risk for acquiring opportunistic infections, reactivating LTBI or exacerbating existing TB among COVID-19 cases.20 In addition, SARS-CoV-2 infection can cause severe immune dysregulation, with a reduction of lymphocyte subsets including CD4 and CD8 T cells, in COVID-19 patients.21,22 Together, the signifcant depletion of CD4 T cells caused by COVID-19 coinfection and the concurrent administration of CSTs for COVID-19 treament might promote reactivation of LTBI.17,23 At present, most COVID-19 cases are not screened or monitored for LTBI before and during immunosuppressive therapy. This situation could delay the diagnosis of TB and increase the number of undiagnosed cases contributing to the overall global TB burden. Therefore, it is important to identify these patients early on and perform LTBI testing during and post-COVID. PHE addressed the need “to improve latent TB infection (LTBI) testing and treatment to prevent reactivation of TB and transmission, a service that has been greatly affected by COVID-19” in its 2020 TB report.6\n\nMassachusetts General Hospital health emergency guidelines recommend a detailed documentation of the TB history of the patient is required for COVID-19 patients and a LTBI reactivation risk mitigation for steroids should be considered when screening high risk patients. As part of the TB elimination strategy, ending the global TB epidemic requires a focus on treating LTBI to prevent future cases and transmission. LTBI reactivation risk mitigation for steroids and immunomodulation could become part of a universal management practice to improve the cascade of TB care. Such a strategy should be shared and considered globally for better management of both TB and COVID-19 cases.\n\n\nA global model to end TB\n\nA global effort has been put in place in order to handle the urgent need to defeat COVID-19 through the “Swiss Cheese” model.24 This metaphor is based on multiple interventions where each intervention (cheese layer) has imperfections (holes in cheese). Due to the “slice gaps”, COVID-19 will always get through. Thus, multiple layers need to be combined in order to block SARS-CoV-2, reduce risk and improve success.24 Such protective layers include social distancing, isolation, mask wearing, contact tracing, vaccinations, and medical care. The Swiss Cheese Model has now been adapted for ending TB and categorises interventions into societal and personal, whilst focusing on a person-centric healthcare system.25 It highlights the need to challenge societal issues that make people vulnerable to TB such as poverty and malnutrition, and emphasises the need for development of better vaccines. TB needs to be de-stigmatised and early care should be encouraged along with personalised quality care for all forms of TB.25\n\nThe WHO has provided key guidance and support for the TB response including maximising remote care by expanding digital technologies, utilising the existing infrastructure and expertise in rapid testing and contact tracing for COVID-19 and expanding it to TB management, ensuring basic infection prevention and control for health staff and patients, providing simultaneous testing for TB and COVID-19 by leveraging TB laboratory networks and platforms; and proactive planning and budgeting for both conditions.7,26 Innovation is the key in re-imagining TB diagnosis and developing an integrated and comprehensive TB care model.26 Further modelling on the COVID-19 impact on TB burden and its determinants could help implement better infection control measures.\n\nTo get back on track with TB progress, we need to have measures and resources to reduce the accumulated pool of undetected people with latent TB. Such measures could be rigorous community engagement and wide-reaching contact tracing to maintain awareness of TB symptoms. The bottom line is that it is crucial to sustain and reinforce the cascade of TB care. Allocating existing TB resources and advocating for additional resources will ease the impact of COVID-19 on the global TB burden. Dealing with COVID-19 is important, but let’s not forget TB, which is meeting us on the other side of this crisis.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nWe would like to thank Dr Andrew Makin, Dr Satwik Kar and Dr Ruth Brignall from Oxford Immunotec, UK for reviewing this paper.\n\n\nReferences\n\nMcQuaid CF, Vassall A, Cohen T, et al.: The impact of COVID-19 on TB: a review of the data. Int. J. Tuberc. Lung Dis. 2021; 25(6): 436–446. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHogan AB, Jewell BL, Sherrard-Smith E, et al.: Potential impact of the COVID-19 pandemic on HIV, tuberculosis, and malaria in low-income and middle-income countries: a modelling study. Lancet Glob. Health. 2020; 8(9): e1132–e1141. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlaziou P: Predicted impact of the COVID-19 pandemic on global tuberculosis deaths in 2020. Epidemiology. 2020. Publisher Full Text\n\nMcQuaid CF, McCreesh N, Read JM, et al.: The potential impact of COVID-19-related disruption on tuberculosis burden. Eur. Respir. J. 2020; 56(2): 2001718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCilloni L, Fu H, Vesga JF, et al.: The potential impact of the COVID-19 pandemic on the tuberculosis epidemic a modelling analysis. EClinicalMedicine. 2020; 28: 100603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organisation: WHO TB report 2020. Geneva, Switzerland: World Health Organisation. Reference Source\n\nTuberculosis in England 2020 report.2020; 191. Reference Source\n\nMcGill International Global Tb Caucus, Briefing note\|Impact of COVID-19 on TB services.2020. Reference Source\n\nTogun T, Kampmann B, Stoker NG, et al.: Anticipating the impact of the COVID-19 pandemic on TB patients and TB control programmes. Ann. Clin. Microbiol. Antimicrob. 2020; 19(1): 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe End TB Strategy: Brochure. Geneva, Switzerland: World Health Organisation; 2015. Accessed on 18 May 2021. Reference Source\n\nProgress towards achieving global tuberculosis targets and implementation of the UN Political Declaration on Tuberculosis. Geneva, Switzerland: World Health Organisation; 2020. Accessed on 18 May 2021. Reference Source\n\nSTOP TB PARTNERSHIP Modelling Report 2020: Accessed on 25 May 2021. Reference Source\n\nSy KTL, Haw NJL, Uy J: Previous and active tuberculosis increases risk of death and prolongs recovery in patients with COVID-19. Infect. Dis. 2020; 1; 52(12): 902–907. PubMed Abstract \| Publisher Full Text\n\nMotta I, Centis R, D’Ambrosio L, et al.: Tuberculosis, COVID-19 and migrants: Preliminary analysis of deaths occurring in 69 patients from two cohorts. Pulmonology. 2020; 26(4): 233–240. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStochino C, Villa S, Zucchi P, et al.: Clinical characteristics of COVID-19 and active tuberculosis co-infection in an Italian reference hospital. Eur. Respir. J. 2020; 56(1): 2001708. Publisher Full Text \| Free Full Text\n\nHuang I, Lim MA, Pranata R: Diabetes mellitus is associated with increased mortality and severity of disease in COVID-19 pneumonia – A systematic review, meta-analysis, and meta-regression. Diabetes Metab. Syndr. Clin. Res. Rev. 2020; 14(4): 395–403. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUdwadia ZF, Vora A, Tripathi AR, et al.: COVID-19 -Tuberculosis interactions: When dark forces collide. Indian J. Tuberc. 2020; 67(4): S155–S162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTham SM, Lim WY, Lee CK, et al.: Four Patients with COVID-19 and Tuberculosis, Singapore, April–May 2020. Emerg. Infect. Dis. 2020; 26(11): 2763–2765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTadolini M, Codecasa LR, García-García J-M, et al.: Active tuberculosis, sequelae and COVID-19 co-infection: first cohort of 49 cases. Eur. Respir. J. 2020; 56(1): 2001398. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGopalaswamy R, Subbian S: Corticosteroids for COVID-19 Therapy: Potential Implications on Tuberculosis. Int. J. Mol. Sci. 2021; 22: 3773. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395: 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang I, Pranata R: Lymphopenia in severe coronavirus disease-2019 (COVID-19): systematic review and meta-analysis. J. Intensive Care. 2020; 8: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhayat M, Fan H, Vali Y: COVID-19 promoting the development of active tuberculosis in a patient with latent tuberculosis infection: A case report. Respir. Med. Case. Rep. 2021; 32: 101344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSiobhan Roberts: The Swiss Cheese Model of Pandemic Defence. The New York Times. Assessed on 25 June 2021. Reference Source\n\nFurin J, Madhukar P: We went all-out to tackle Covid-19-TB needs the same approach. The Telegraph. Assessed on 25 June 2021. Reference Source\n\nRuhwald M, Carmona S, Pai M: Learning from COVID-19 to reimagine tuberculosis diagnosis. Lancet Microbe. 2021; 2(5): e169–e170. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "209613", "date": "23 Oct 2023", "name": "Ponlagrit Kumwichar", "expertise": [ "Reviewer Expertise Tuberculosis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRegarding the paper titled \"All eyes on COVID-19, let's not forget Tuberculosis,\" I believe a major revision is imperative. My comments are as follows:\nThe introduction's portrayal of TB seems outdated. TB is now understood as a spectrum disease; hence, this section requires an update.\nThe discussion concerning the impact of COVID-19 on TB services appears superficial. I recommend a more extensive literature review, including studies from various countries. It would be beneficial to amalgamate evidence from lower-, middle-, and upper-income nations comprehensively.\nIn the section \"Are we meeting the ambitious targets of the WHO End TB Strategy?\" the authors should explore the possibility of underreporting of TB cases. Overburdened healthcare workers, especially in LMIC countries with weaker medical database systems, might overlook reporting some cases.\nRegarding the \"Cursed duet\" segment, I suggest the authors detail their findings more explicitly, using objective metrics like Pulmonary function tests.\nFor the \"From LTBI to active TB\" section, the authors should reference more recent evidence. Few observational studies have been published since 2023 that could provide valuable insights.\nA significant omission is the lack of emphasis on TB treatment monitoring. Elements like drug adherence, side effect surveillance, and post-TB rehabilitation are crucial for TB elimination. Even with advanced lab detection tests and drugs, neglecting patient care could lead to a surge in MDR TB cases.\nThe paper seems to readily accept new drugs and vaccines without adequately addressing potential long-term side effects, which remain largely unknown.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No", "responses": [] }, { "id": "223419", "date": "28 Nov 2023", "name": "Katherine Robsky", "expertise": [ "Reviewer Expertise Epidemiology of 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\nThis opinion piece lays out the potential impact of the COVID-19 pandemic on TB. This is indeed an important issue, and even as COVID-19 restrictions have been relaxed, it is important to take steps to address some of these consequences. However, the argument of the paper is a bit muddled and in some places not supported by the evidence.\nThe focus of the article is unclear. The authors cite examples from high-income, low-TB burden countries (the UK) and low-income, high-TB-burden countries without providing clear context on why the outcomes might be different in these very different settings.\n\nSome of the conclusions drawn about the impact of COVID-19 on TB are not supported by the literature: for example, the authors cite data from 2018-2019 as evidence of COVID-19 as a setback, but these challenges pre-date the COVID-19 pandemic\n\nThe underlying causes of some of these impacts are not clearly explained. Changes in movement restriction, funding, policies, healthcare workforce, and other upstream contexts lead to the observed impacts on health care delivery. This is not explored in the paper but will be important to understand as the authors advocate for getting “back on track” with TB progress.\n\nThe authors don’t discuss how many interventions – masking, contact investigation, social distancing, improving air ventilation – can prevent both COVID-19 and TB. Therefore, there are many opportunities in which investing in the prevention of one disease can hep control the other.\n\nThe authors don’t really offer any suggestions for getting TB back on track. Some studies have shown that intensive active case finding conducted in the few years after movement restrictions may have a big impact on long term TB trends. These extra activities are likely critical for improving TB control. The authors discuss the expansion of infrastructure and contact tracing for COVID-19 to TB, but in fact that infrastructure was largely borrowed from TB and repurposed for COVID\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "223417", "date": "28 Nov 2023", "name": "Alex Zimmer", "expertise": [ "Reviewer Expertise epidemiology", "tuberculosis", "global health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for submitting your manuscript for review. After careful consideration, I regret to inform you that I cannot recommend it for Indexing in its current form. My decision is based on the fact that the article presents information that seems outdated in light of the ongoing developments in the field of tuberculosis (TB) and the COVID-19 pandemic. The references and data predominantly focus on the situation as it was 1-2 years ago, which does not reflect the current state of TB management, especially in the context of recent progress made post-COVID. As a whole, the article does not contribute novel information to the literature.\nMinor comments\nThere is a noticeable discrepancy between the title and the abstract of the paper. The title suggests a comprehensive approach to active TB, whereas the abstract predominantly focuses on latent TB infection (LTBI). To maintain consistency, I suggest either narrowing the focus in the abstract or revising the title to more accurately reflect the LTBI emphasis.\n\nThe reference to a \"successful TB control programme\" is vague and requires clarification. The assertion of TB being successfully controlled is questionable, given the current challenges in the field.\n\nThe manuscript states \"We are now more than one year [into the pandemic]\" which is outdated. We are, in fact, well into the third year, and the landscape of TB care has evolved significantly, as evidenced in the latest Global TB Report (2023).\n\nThe paper's oversimplification of TB infection into two phases is contrary to recent evidence suggesting a spectrum-based understanding. I recommend revisiting the current literature for a more nuanced perspective, such as the study referenced in DOI: 10.1183/20734735.0079-2021.\n\nThe introduction lacks comprehensive context regarding LTBI, particularly in relation to COVID-19's impact on LTBI treatment and outcomes. Expanding this section with prior literature would strengthen the manuscript.\n\nThe section on the impact of COVID-19 on TB services generalizes the discussion about TB, whereas a more concentrated examination of LTBI would be beneficial.\n\nThe assertion that COVID-19 will dramatically set back TB progress is already well-established in previous Global TB Reports. The manuscript, as it stands, seems to reiterate these findings without adding new insights to the literature.\n\nThe application of the Swiss Cheese model in the context of TB has been previously discussed in the literature (e.g., DOI: 10.1136/jech-2021-217529). If you wish to utilize this model, proper citation and comparison with existing work are necessary.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "205253", "date": "14 Sep 2024", "name": "Shamsuddeen Yusuf Ma'aruf", "expertise": [ "Reviewer Expertise Tuberculosis", "Nanoparticles", "Vaccine R&D", "Immunology", "Bioprocessing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author should revisit the title of the paper. Instead of \"All eyes on COVID-29, Let's not forget Tuberculosis\", the authors can use \"The status of TB Control, in the face of emerging infectious disease\".\nThe authors should revisit the citations. The reference of WHO report on TB data is outdated - WHO recently released updated TB data. This is similar to the author-cited papers. It will be ideal if the authors look into recent articles (e.g., Ma'aruf et al., 20231).\nThe impact of COVID-19 is immense, however success has been achieved in controlling and managing COVID-19. Hence, the impact of this paper is partial, but the authors could improve the paper by referring to past limitations and emphasis on success achieved and how to avoid the decline/setback in TB control in future pandemics.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-158
https://f1000research.com/articles/10-751/v1	04 Aug 21	{ "type": "Research Article", "title": "Trap of weights: The reuse of weights in the floating catchment area (FCA) methods to measuring accessibility", "authors": [ "Lina Zhang" ], "abstract": "Background: Geographic weights are vital in the floating catchment area (FCA) method of accessibility measurements due to their simulation of spatial barriers in various ways. When modelling population demand, geographical weights with different distance decay coefficients can reflect diverse distance tolerances in facility utilization and could lead to erratic accessibility results. Quantifying accessibility as the sum of weighted supply-demand ratios can alleviate the distance decay coefficient's influence and generate stable geographic patterns. However, the effects of weighted ratios on different FCA models and resources have not been investigated. Methods: To identify impacts of weighted ratios on various FCA variants, this study contrasted the accessibility calculated from the sum of ratios (access) and the sum of weighted ratios (access ratios) within three prevalent FCA models: enhanced two-step FCA (E2SFCA), modified two-step FCA (M2SFCA), and three-step FCA (3SFCA). In addition, the accessibilities of various resources evaluate the stability of the weighted ratios' effect. This study therefore examined the accessibilities to primary schools, job opportunities, and major hospitals in Shanghai. Shanghai is a case study that provides lessons on using big data to measure accessibility in metropolitan areas. Results: Geographic weights can not only mitigate the impact of the distance decay coefficients, but can also eliminate model features, which reduces the performance of the M2SFCA's supply decay and the 3SFCA's population demand adjustment in accessibility results. Moreover, weighted ratios tend to overestimate accessibility in marginal communities that lie within fewer catchments, regardless of the resource type. This tendency can lead to an epistemological trap that creates an inaccurate and counter-intuitive perception of resource distribution in a given area. Conclusions: The results identify a gap between the methodological logic and the empirical perception in accessibility measurements. This study concludes that the use of geographic weights needs to be cautious and epistemologically consistent.", "keywords": [ "Spatial accessibility", "geographic weights", "E2SFCA", "M2SFCA", "3SFCA" ], "content": "Introduction\n\nAccessibility is a fundamental indicator for detecting spatial equity in distributive justice (Talen, 2001). Derived from Rawls’s philosophy of justice, distributive justice offers deontological guidance on distributions of benefits and burdens across members of the society (Lamont, 2017; Rawls, 1971). Distributive justice is a classic and basic theme (Arrow, 1973), as distributions of benefits and burdens have a substantial impact on people’s lives and social inequalities. In essence, distributive justice includes guidance for political processes and structures, while spatial equity more specifically examines the social impact of the physical distribution of certain goods and services (Dadashpoor et al., 2016; Taleai et al., 2014). Accessibility describes the distributive supply-demand relationship of resources and can be visualised using various geographic information system (GIS) models (Higgs, 2004; Luo and Wang, 2003). Accurate measurements of accessibility are crucial for identifying areas of poor resource distribution (Yang et al., 2006), revealing spatial reflections of social inequality (Guagliardo, 2004), and providing policymakers with practical suggestions on how to achieve distributive justice (Neutens, 2015).\n\nIt is important to consider methodologies in accessibility measurements (Baradaran and Ramjerdi, 2001; Bell et al., 2013; Sherman et al., 2005). On one hand, the use of different models, which yield divergent outcomes, indicates that there are heterogeneous understandings of accessibility (Bunel and Tovar, 2013; Guagliardo, 2004). For example, the buffer model (Nicholls, 2001) defines accessibility in terms of facilities’ service areas, which are measured by the radial distance from a given facility, while the isochronic model (Cervero et al., 1998) uses the travel time to the facilities as the main indicator of accessibility. The gravity model originally defined accessibility within a census tract as the number of facilities divided by the distance between the facilities and the census tract (Hansen, 1959). Weibull (1976) later added population demand to the denominator to simulate the competition among residents for limited capacity services. On other hand, manifold factors in GIS models can affect the distributive pattern of accessibility. These factors include spatial scale (Higgs, 2004), transport model (Mao and Nekorchuk, 2013; Ni et al., 2019), unit aggregation (Apparicio et al., 2008; Hewko et al., 2002), and distance decay function (Kwan, 1998).\n\nFloating catchment area (FCA) metrics have been among the most widely applied models in accessibility measurements (Neutens, 2015). Based on service area, FCA metrics utilize the catchment concept to describe the efficacy area of a location, including the possible geographical area that describes resident activities for a given community. In FCA methods, accessibility within a census tract is defined as the sum of the facilities’ supply-demand ratios within its catchment (Luo and Wang, 2003). A strength of FCA metrics is that it combines the advantages of the gravity model and the cumulative-opportunity model. The gravity model provides a global distribution of accessibility by considering supply and consumer consumption in each area (Geertman and Ritsema Van Eck, 1995), whereas the cumulative-opportunity model measures spatial equity in terms of potential opportunity. This combination drives the flexibility of FCA methods. Scholars have endeavoured to create various FCA variants for accurate simulation of accessibility, including the enhanced two-step FCA (E2SFCA) (Luo and Qi, 2009), the modified two-step FCA (M2SFCA) (Delamater, 2013), the three-step FCA (3SFCA) (Wan et al., 2012b), and the flow-based 2SFCA method (Tang et al., 2017).\n\nDistance weights play an important role in FCA metrics development. The E2SFCA method applies distance weights to simulate people’s declining intent to facility utilization as the distance to them increases. Similarly, the M2SFCA considers the decrease in service capacity with increasing distance, by attaching weights in supply calculation. The 3SFCA calculates the share of distance weights in each facility to simulate the competition among suppliers. However, when applying weight formulas to represent the population demand, the value of the decay coefficient can significantly alter accessibility distribution (Giles-Cortia and Donovan, 2002). This is because the coefficient can sensitively change the population demand and accessibility is calculated as the aggregation of supply-demand ratios (Bauer and Groneberg, 2016). To reduce the sensitivity of the decay coefficient, Wan et al. (2012a) argue that multiplying weights by the original supply-demand ratio can generate a relatively stable accessibility result. This repeated use of distance weights, so-called ’the reuse of weights’, simulates spatial barriers in service capacity, population demand, and supply-demand ratios; thus, it has become a common component in FCA methods.\n\nHowever, taking accessibility as the sum of weighted ratios and examining its impact on various FCA models has not yet been performed. This improved method transforms the accessibility from a sum of opportunities to an accumulation of weighted ones. Using this method also raises the question of the extent to which weighted ratios provide stability in different FCA variants. Wan et al. (2012a) demonstrate how weighted ratios provide resistance to varying values of the distance decay coefficient in the E2SFCA, while this conceptual transformation could lead to stability across FCA models and potential fallacies. To systematically measure the impact of reusing weights, this study uses three prevalent FCA models (i.e. E2SFCA, M2SFCA, and 3SFCA) to compare accessibility as the sum of ratios and accessibility as the sum of weighted ratios. For clarity, this study defines the original non-weighted accessibility as ’access’ and the weighted ratios as ’access ratios’. In addition, this research uses Shanghai as a case study to provide accessibility measurements based on big data and metropolitan case evidence. Shanghai is one of the most populated metropolises in the People’s Republic of China, with a population of 24.20 million in 2017 (Shanghai Statistics Bureau, 2018). During its rapid urbanisation, social inequality has been a severe problem (Li and Wu, 2008), and accessibility measurements are necessary for its distributive justice and sustainable development. Based on point of interest (POI) data from 2017, this study compares the accessibility of three resources in Shanghai, namely primary schools, job opportunities, and major hospitals. By comparing the impact of reusing weights on different FCA models and resources, the study identifies the trap of weights, provides empirical evidence for the gap between methodology and epistemology, and justifies the importance of epistemological criteria in the evaluation of accessibility methods.\n\n\nReusing weights in accessibility measurements\n\nThe complexity in modelling spatial accessibility is derived from its abundant definitions and methodological flexibility, which vary across different fields and build distinct taxonomies. For example, in transportation, accessibility is defined as the potential to reach spatially dispersed opportunities, with a focus on travel cost and transport policy (Páez et al., 2012). In health studies, accessibility of medical facilities is one of the five dimensions (accessibility, availability, accommodation, affordability, and acceptability) that describe the relationship between health facilities and patient utilization (Penchansky and Thomas, 1981). Guagliardo (2004) deconstructs the concept of health accessibility into two stages (potential and realized) and two dimensions (spatial and aspatial). The former refers to the difference between potential opportunity simulation and actual utilization, while the latter considers spatial factors and socioeconomic attributes.\n\nThe many definitions of ‘accessibility’ lead to a variety of accessibility-modelling methodologies. The initial container method takes accessibility as the number of facilities in a given unit (Talen and Anselin, 1998). The minimum distance and travel cost methods prioritise transportation in determining facility utilization (Guy, 1983). The Kernel density method estimates the relationship between facility density and population density (Yang et al., 2006). However, few models consider user behaviour in their measurements. For example, residents’ facility utilization can extend beyond their communities, and has a declining frequency as travel distance increases (Higgs, 2004). This is due to so-called ‘spatial barriers’ (Guagliardo et al., 2004; Neutens, 2015). To account for this, the gravity model introduces a distance decay function in assessing population demand and defines accessibility as the sum of facilities’ supply-demand ratios related to a given community. FCA methods are special applications of the gravity model that use catchment areas to simulate the geographical sphere of facility utilization and calculate the supply-demand ratios within catchments (Luo and Wang, 2003).\n\nNumerous factors in GIS models affect the distributive patterns of accessibility maps. Different unit aggregations can generate divergent distributions because of the modifiable areal unit problem (MAUP) (Bell et al., 2013; Dadashpoor et al., 2016). As a long-standing statistical bias, the MAUP refers to the fact that different shapes and sizes of the unit can alter the geographic results (Hewko et al., 2002; Zhang et al., 2011). Bell et al. (2013) contrasted accessibility in neighbourhood and census tract to highlight the social characteristic of the unit aggregation. Additional confounding includes the methods of calculating distance, transport mode, catchment size, and facility capacity (Ni et al., 2019; Pan et al., 2018; Wan et al., 2012b). Apparicio et al. (2008) conducted a comprehensive comparison to examine the influence of different distance types and units aggregation. Geographic weights also play an important role in the accessibility measurements, as illustrated in the next section.\n\nAs this study aims to examine the influence of reusing weights in FCA methods, the complexity of modelling accessibility increases the difficulty of selecting appropriate evaluation dimensions for their impact. Despite the mentioned influencers, the study chose two dimensions—model type and resources—as the evaluation dimensions for the impact of the reusing weights. Bunel and Tovar (2013) identified the model type as a key issue that can generate different empirical results in accessibility measurements. Although the methodological improvements have driven the reuse of weights, it is a conceptual transform in the FCA methods (Wan et al., 2012a). This conceptual shift should be investigated in a variety of FCA variants for its legitimacy. Moreover, the FCA methods are applied in measuring various resources’ accessibility, including health facilities, jobs, and urban parks (Delamater et al., 2019; Kawabata and Takahashi, 2005; Xie et al., 2018). The wide application of the FCA methods to different resources reflects the fact that the rationality of the methodology can transcend the resource characteristics. This means that differences in resource types do not affect the validity of the method. Thus, comparing the same method across resources allows further testing of the methodological generality. This study introduces a dimension of model type to test the legitimacy of reusing weights and a dimension of resources to examine its generality. The following section reviews how methodological improvements have driven the reuse of weights in FCA methods, as well as the potential limitations of reusing weights.\n\nDistance weights play a major role in the development of FCA methods (Langford et al., 2012). A series of methodological improvements have focused on changes in the application of weights for varied purposes (Luo and Qi, 2009; Wan et al., 2012a,b). The original 2SFCA (Luo and Wang, 2003) has two calculation stages: first, the population demand within the supplier’s catchment area is summed to calculate the supply-demand ratio for each supplier. Second, these supply-demand ratios are then summed within the catchment based on the neighbour tracts, which is the value of access (see Equation (1)).\n\nAi2S represents the access at tract i based on the 2SFCA method, Ci is the catchment centred at tract i, Rj is the supply-demand ratio of supplier j which falls in the catchment centred at tract i, Sj is the supply capacity of supplier j, Cj is the catchment centred at supplier j, and Dk is the population demand of tract k, which falls in the catchment centred at supplier j.\n\nThe E2SFCA method enhances the stimulation of population demand in 2SFCA (Luo and Qi, 2009). It applies a distance decay function in the population demand to change it from a dichotomous calculation to a continuous variable. Equation (2b) is the distance decay function in the form of a power function. The distance weight and population demand decline as the distance increases.\n\nAiE2 represents the access calculated by the E2SFCA method, RjE2 is the supply-demand ratio of supplier j which falls in the catchment centred at tract i calculated by the E2SFCA method, wkj is the distance weight between tract k and supplier j, dkj is the distance between tract k and supplier j, β is the decay coefficient, and the other variables are the same as in equation (1).\n\nFurthermore, the M2SFCA method argues that not only population demand decreases with distance, but also service effectiveness of suppliers (Delamater, 2013). In M2SFCA, the effectiveness is calculated using distance weights plus the supply capacity for each pair of tracts and suppliers (see Equation (3a)). AiM2 represents the access at tract i based on the M2SFCA method, wij is the distance weight between tract i and supplier j. Equation (3b) is the distance decay function, dij is the distance between tract i and supplier j, and the other variables are the same as in Equation (2a) and (2b).\n\nUnlike M2SFCA, the 3SFCA method tries to solve the exaggeration of population demand in the E2SFCA (Wan et al., 2012b). As in Equation (2a), the population of tract k is calculated multiple times in accordance with the number of suppliers within tract k’s catchment. The solution is to introduce the supplier weights into population demand. For certain tract k, its supplier weight of supplier j (wkjS) equals its distance weights wkj divided by the sum of the distance weights of all suppliers within its catchment. In Equation (4a), Ai3S represents the access at tract i based on the 3SFCA method, wkj is the distance weight between tract k and supplier j. Equation (4b) is the calculation of supplier weight wkjS between tract k and supplier j, where supplier m is the supplier’s fall in the catchment of tract k, and wkm is the distance weights between tract k and supplier m.\n\nFigure 1 shows the development of four FCA methods. For various intents, the adaptation of geographic weights has continued throughout the FCA improvement process. Based on the original 2SFCA, the E2SFCA introduces distance weights in population demand to model spatial barriers in resource utilization, which results in declining population demand as the distance between the supplier and the consumer increases. Further to this, the M2SFCA has placed distance weights on the supply capacity to model distance barriers in resource effectiveness. The 3SFCA improves on the E2SFCA’s population demand calculation by factoring in the supplier weight. For a distance between a census unit and a supplier, its supplier weight equals its distance weight divided by the sum of the distance weights of all suppliers that are located in the catchment area of the unit. The supplier weight distributes population demand in accordance with spatial barriers and simulates the competition among suppliers. Especially in the M2SFCA and the 3SFCA, the reuse of weights is the main method to achieve their improvement goals.\n\nThe adaptation of geographic weights has continued throughout the improvements.\n\nSimilarly, geographic weights are the solution to the high sensitivity of the distance decay coefficient, which is a common problem in the methods described above (Xing et al., 2018). The value of the distance decay coefficient β (see Equation (2b),(3b), and (4b)) can significantly change the accessibility distribution, even from a monocentric to a decentralised structure. This raises two questions: how should a suitable value for the decay coefficient be determined, and to what extent should it influence the accessibility distribution? For the first question, the normal arbitrary value of β ranges from 1.0 to 2.2 (Luo and Wang, 2003; Luo and Qi, 2009). One practical and rationale way to determine the coefficient values for the different facilities is to use questionnaires to simulate the spatial barriers in facility utilization (Giles-Cortia and Donovan, 2002). For the second question, Wan et al. (2012a) argue that accessibility distribution should remain stable regardless of the coefficient value. This is because the decay coefficient describes how utilization intention and service effectiveness declines with increasing distance. For example, when β equals 1, the distance weight for a census tract 5 km from the hospital equals 0.2, while when β is 2, the weight equals 0.04 (see Equation (2b)). Both values can be appropriate according to divergent individual preferences. However, it is difficult to deal with the uncertainty around the individual use of facilities in the place-based FCA methods. Their solution is to retain the distance-decaying nature, but to remove the influence of β by taking accessibility as the sum of weighted supply-demand ratios, i.e. the access ratio. The distribution of access ratio is relatively stable, with similar patterns under different values of the distance decay coefficient. Table 1 shows the comparison of access (Ai) and access ratio (AiR) of the three methods. In the E2SFCA and the M2SFCA, the access ratios equal the original value multiplied by the distance weight. Whereas in the 3SFCA, it is the original value multiplied by the supplier weight and the distance weight.\n\nModel formulas of access (Ai) and access ratio (AiR), where E2SFCA is the enhanced two-step floating catchment area (FCA), M2SFCA is the modified two-step FCA, and 3SFCA is the three-step FCA.\n\n* Ci is the catchment centred at tract i. RjE2 is the supply-demand ratio of supplier j which falls in the catchment centred at tract i calculated by the E2SFCA method. The supply-demand ratios calculated by the other two methods are similar.\n\n** wij is the distance weight between tract i and supplier j. wijS is the supplier weight between tract i and supplier j.\n\nDefining accessibility as the sum of weighted ratios has been widely applied in various FCA methods due to its relatively simple implementation (Fransen et al., 2015). In the measurements of access ratios (AiR), there is limited consideration of the decay coefficient value or the stability of the accessibility distribution. However, this weighted ratio has not been examined for its impact on the FCA variants. There is a potential misidentification of the ‘omnipotence’ of weights in FCA methods. For example, reused weights in population demand, which is the population multiplied by the supplier weight and the distance weight in the 3SFCA, are not equivalent to the row normalisation of the previous population (see Figure 2). The next section discusses the possible side effects of reusing weights in FCA methods.\n\nComparison of accessibility values as the sum of supply-demand ratios (Ai) and the sum of weighted supply-demand ratios (ARi). ARi enlarges the resource acquisition of grids closer to supplier’s location.\n\nAlthough researchers have discovered several limitations of FCA methods, the reuse of weights has not gained sufficient attention. Among those identified limitations (i.e. catchment sizes, overestimation of population, and the MAUP), the application of weights focuses on the value of the distance decay coefficient and the function type of distance decay (McGrail, 2012; Bunel and Tovar, 2013). Evidently, the impacts of distance weights have been simplified to only consider the differences between decay coefficient β values and common forms of distance decay, including the exponential function, the inverse-power function, and the Gaussian function (Kwan, 1998; Neutens, 2015).\n\nHowever, the manipulation of weights can also have a substantial impact on modelling accessibility. Delamater (2013) has discovered one side effect of reusing weights in the 3SFCA. He modelled a simple dynamic topology that gradually moved one far-away unit closer to a facility-adjacent area. During this process, the 2SFCA method calculates the accessibility for this unit as monotonically increasing, while the 3SFCA method calculates the accessibility as first decreasing and then increasing. This is because reusing weights in population demand, which considers supplier weights and distance weights simultaneously, generates a non-monotonically varying distance decay function in the 3SFCA. Although, the non-monotonic variation in the 3SFCA may have a limited impact in complex topologies.\n\nThe use of weighted ratios can produce additional problematic results. Figure 2 illustrates one side effect of defining accessibility as the sum of weighted ratios. The grey quadrilaterals are census tracts and the red pentagrams are supplier locations. To simplify the calculation, this example applies the E2SFCA method and a piecewise function as the distance decay function. The weights of zone 1, zone 2, and zone 3 equal 1, 0.75, and 0.5, respectively. The first step calculates the supply-demand ratio of each supplier (S1 and S2), which is the weighted population of tracts divided by the suppliers’ capacity. The second step aggregates the supply-demand ratio of suppliers within the catchment of each grid centroid. Detailed calculations are provided below:\n\nRSn: the supply-demand ratio of supplier Sn.\n\nAGn: the access of grid Gn (accessibility as the sum of supple-demand ratios).\n\nAGnR: the access ratio of grid Gn (accessibility as the sum of weighted ratios).\n\nAs shown in Figure 2, the access (Ai) of Grid 1 is greater than that of Grid 2, while their access ratios (AiR) are reversed. Grid 1 has a reduced access ratio because of its location within two suppliers’ Z2 catchments, while Grid 2 lies within only one supplier’s Z1 catchment. Defining accessibility as the sum of weighted ratios may exaggerate a community’s resource consumption when it lies in close proximity to suppliers. In this instance, it remains uncertain which census tract contains higher service capacities. Still, this example reveals that access ratios exaggerate the accessibility of population units with closer proximities to suppliers and, therefore, greater geographic weights.\n\nThe exaggeration of the access ratio in units with closer proximities to amenities should be examined in the context of complicated topologies. There remains the possibility that this exaggeration is either imperative for the precise description of resource distribution, or irrelevant in the context of complex geography. Moreover, how this distortion of the access ratio relates to the modelling approach and the type of resource in question needs to be further investigated. Access ratios produced by various applications of distance weights in the FCA methods could alter its impact on accessibility results. Furthermore, due to their diverse topologies, varied resources with distinct differences in user behaviours appropriate to the size and attributes of their catchments may also influence the effect of weighted ratios. As mentioned above, there are two levels of weighting applications. The differential applications of geographical weights in supply and demand simulations shape FCA variants, while the access ratio helps quantify accessibility. Accordingly, this study focuses on the impact of the access ratio and attempts to build a systematic evaluation by comparing Ai and AiR, with consideration of the aforementioned influencing factors.\n\n\nMethods\n\nAs a global centre for finance, innovation, and transportation, Shanghai is one of the most populated metropolises in the People’s Republic of China. Its rapid urban expansion has caused a gap in distributing various resources between urban and rural areas (Xiao et al., 2017). Accessibility measurements can detect the spatial equity of various resources in the city’s urban development. Moreover, taking Shanghai as a case study provides an empirical analysis of accessibility models in complex topology and a large city. This study chose Shanghai’s administrative land region (6340.5 km2) as the study area.\n\nThe original data was purchased from Urban Data Party (UDP) via its data service (please see Underlying data). The datasets provided by UDP are accessed from AMAP through web crawler technology. The Urban Data Party membership provides cell phone signalling data, road network data, and the points of primary schools, companies, and hospitals in Shanghai in 2017. This study used the cell phone signalling data to generate the Shanghai population grid. UDP has subsequently deleted the 2017 cell phone signalling data, and instead cooperated with WorldPop et al. (2017) to provide a broader map of population density that is publicly available.\n\nAs an alternative to the restricted UDP datasets, OpenStreetMap provides publicly available POIs and road network data of Shanghai, while WorldPop provides the population density of Shanghai (please see Underlying data for more information).\n\nThis study examines the role of weighted ratios in accessibility measurements from two dimensions. The study is based on the contrast between access (Ai) and access ratios (AiR) , as shown in Table 1. The first dimension is the different FCA metrics. The E2SFCA, M2SFCA, and 3SFCA models were chosen because of their prevalence and multiple weight applications with various aims. Methodological comparisons assess whether the impacts of access ratios change across different models. Therefore, the purpose of this study is to examine the relationship between the type of FCA model and the side effects of reusing geographic weights.\n\nThe second dimension is the impact of access ratios for different types of resources, including primary schools, jobs opportunities, and major hospitals. These empirical comparisons evaluate how access ratios perform in realistic and complex topology.\n\nTable 2 shows the detailed characteristics of the three objects, including their catchment areas and spatial distributions. Primary schools are distributed dispersedly in accordance with neighbourhood locations, as pupils have limited capabilities for long-distance travel, and will therefore usually attend the school closest to their residence. In contrast, jobs and major hospitals are distributed in a centralized way, as workers and patients can typically endure longer distances to obtain income and medical services. Furthermore, healthcare-related behaviours are more tolerant to increased distance than work-related commuting behaviours because obtaining healthcare is typically a matter of necessity and occurs less frequently. Even with the same distance radius, the catchments of hospitals involve more flexible behaviours than those of job opportunities. Therefore, primary schools have a catchment radius of three kilometres for their limited-service areas, while job opportunities and major hospitals both have a 20 kilometre radius.\n\nThere are three stages of data analysis: data pre-processing, generation of the distance matrices and the calculation stage. The first pre-processing stage provides the basic datasets for the distance matrices. The distance matrices refer to the network distance between each population grid and each POI. The calculation stage computes the values of access and access ratios for each grid.\n\nThe first stage processes the population grid and the POI datasets in the software Quantum Geographic Informtion System (QGIS 3.16.6). This study first generated the population density from the 2017 cell phone signalling data for the population grid. It then used the default Zonal Statistics plug-in in QGIS to transfer the population density into a 250250 m population grid (28,037 units) in conjunction with the Statistical Yearbook data (Shanghai Statistics Bureau, 2018). For the POIs downloaded from UDP (or OpenStreetMap), it is necessary to delete wrong and duplicate points in the QGIS. Specifically, the number of companies (over two hundred thousands points) is too large to generate distance metrics in QGIS. Therefore, the job opportunities are estimated in the 250250 m grid by multiplying the company size and the number of companies. Major hospitals are those whose official level is greater than Grade II. All service capacities are calculated as the number of objects. For datasets downloaded from OpenStreetMap and WorldPop, it is crucial to ensure they have the same coordinate systems and projections.\n\nThe second stage is to form the distance matrices between population grids and POIs datasets. The QNEAT3 plug-in in QGIS generated the distance matrices between the population grid’s geometric centre points and the POIs based on the road networks. In this study, the distance is computed in the car-drive mode.\n\nBased on the distance matrices, the third stage calculates the spatial accessibilities to three types of facilities within three FCA models. Python (3.7.0) is used to calculate the accessibility of each population grid in three existing models (2SFCA, M2SFCA, and 3SFCA). Using the custom codes available in Extended data (Zhang, 2021) can realise the calculations of access (Ai) and access ratios (AiR) in the three models. An alternative is to reference the codes of access (Ai) in 2SFCA and 3SFCA models at the spatial_access package. This package requires strict forms of original data and generates limited accessibility results.\n\n\nResults\n\nAs a continuation of the previous study design outlined in the Methods, the analysis examines methodological and empirical implications of the results by comparison of Ai and AiR. Figure 3 presents the access results (Ai), meanwhile, Figure 4 presents the results of access ratios (AiR). In Figure 3, the three FCA metrics generate significantly different spatial structures, whereas Figure 4 shows similar structure of access ratios for the resources.\n\n(A) enhanced two-step FCA (E2SFCA), (B) modified two-step FCA (M2SFCA), and (C) three-step FCA (3SFCA).\n\nThree floating catchement area (FCA) metrics generate similar spatial distribution of accessibility according to resources. (A) enhanced two-step FCA (E2SFCA), (B) modified two-step FCA (M2SFCA), and (C) three-step FCA (3SFCA).\n\nFrom a methodological perspective, weighted ratios produce stable accessibility patterns regardless of the FCA model type. Compared to the non-weighted maps (Figure 3), weighted ratios (Figure 4) result in similar global spatial structures of accessibility distribution. The weighted ratios produce stable patterns not only under different values of the distance decay coefficient (Wan et al., 2012a), but also under various FCA methods. Particularly for resources with a low distance tolerance, such as primary schools, weighted ratios produce maximum accessibility to the same locations. As the tolerance of user behaviour increases, the nuances between the three methods become more pronounced. The 3SFCA creates more local differences in global structure between job accessibility and health accessibility compared to E2SFCA and M2SFCA. This implies that user behaviours in resource utilization and the aggregation of original POIs can affect the stability of the weighted ratios.\n\nOne possible explanation for this pattern stability is the side effect of weighted ratios shown in Figure 2. Weighted ratios overestimate the accessibility to resources of communities with high distance weights and a close proximity to suppliers, as higher access ratios are observed in peripheral areas rather than city centres (see Figure 4). Units in peripheral areas tend to have fewer suppliers in their catchments, resulting in high supplier weights. For those units with only one facility and close proximity to it, their access ratios (AiR) are almost the same as their access values (Ai). Conversely, units in the city centre with multiple suppliers have access ratios lower than their access values because of their relative small distance weights multiplied several times. Therefore, access ratios of units with fewer suppliers in the periphery are relatively enlarged.\n\nHowever, there remains a common point between weighted and non-weighted ratios. Both results show that the accessibility distribution differs depending on the type of amenity. It is logical that accessibility structures for primary schools, jobs, and main hospitals are dissimilar, as each object has its own distinguishing distribution. This suggests that the accessibility maps have an evidence-based foundation in addition to methodological influences.\n\nAlthough weighted ratios eliminate the effect of the decay coefficient values, the validity of model stability across model types remains contentious. In Figure 4, the nuances between three FCA methods exist in partial districts and at the local level. Those subtle differences make it difficult to verify the intended characteristics of FCA methods and further lead to the dispute over the necessity of improvements. On the contrary, Figure 3 visualises the different model improvements in results. The improvement of the M2SFCA, that is, the distance decay of service effectiveness, converts the uniform value of accessibility into a gradual value (especially A3 and B3 in Figure 3). The enhancement of the 3SFCA, which models demand division among suppliers, reduces the overestimation of the population in the facility cluster and produces a more concentrated distribution (see B1, C1, B2 and C2 in Figure 3). On the other hand, it could also be considered a methodological advantage of weighted ratios to create stability across FCA methods. To determine which calculation, using either non-weighted or weighted ratios, is rational, one possible approach is to apply the evidence-based foundation and introduce empirical judgements.\n\nAlthough their results vary, both calculations of access and access ratios are supported by methodological rubrics. Despite these methodological rationalisations, the variability of results leads to diametrically opposed empirical judgements. These contrasting discernments could make it easier to choose a more rational method, based on experiences. Therefore, the evidence-based foundation of accessibility measures may also provide empirical criteria for the choice between Ai and AiR, by linking the mapped results with common sense.\n\nWhile there are varying degrees of variability between access and access ratios for different resources, they show a common characteristic among the resources. Maps of access ratios (AiR in Figure 4) share similar structures with those of the access calculated by the M2FCA (Ai B-maps in Figure 3). This is due to the reuse of weights in the M2SFCA, as described in the formulas shown in Table 1. Furthermore, the reused weights divide accessibility distribution into two kinds of judgements. In the case of primary schools, the access ratios in Figure 4 indicate that the central city contains a shortage of primary education resources, while the north-eastern island (Chongming Island) has the greatest abundance of those resources. However, the 3SFCA method in Figure 3 concludes the opposite: that the city centre is the most resource-rich, while Chongming Island lacks basic education facilities. Similarly opposed conclusions are the polycentric and monocentric structures of accessibility to job opportunities and major hospitals generated from Ai and AiR.\n\nThe difficulty in judging the validity of opposing results lies in the ratio attribute of accessibility. The provision of services and the needs of the population are reflected in visual and definitive indicators of their quantity. Each facility points has certain indicator of its service capacity, such as the number of physicians in a hospital. Each residential unit obtains an accurate number for its population. However, the relationship between supply and demand is indirect, uncertain, and dynamic. The maximum supply-demand ratio could occur in the suburbs or the city centre based on empiricism.\n\nOne possible solution is to apply Kantian ‘a priori knowledge’, which combines empiricism and rationalism. First, key locations are contrasted to provide actual and intuitive measurements. Field research on the key locations provide empirical evidences for the resource distribution. Second, the overall judgements of resource distributions in previous studies are discussed to choose the most accurate global structure of accessibility. These previous studies provide the ‘a priori knowledge’ of resource distributions based on actual utilizations.\n\nFieldwork may provide insight into the comparison of the accessibility values of key points and thus inform model recommendations. In accessibility to primary schools, the catchment of School A at Chongming Island (see (a1) in Figure 4) has one of the highest access ratios AiR, although the island is famous for its agricultural products and natural landscapes. School B is one of the medium values also located on the island. Field research reveals that School A serves only its collective farm, which is a socialist legacy with limited productivity and a decreasing population. School B, on the other hand, is located in a more densely populated area and provides a higher level of educational resources for the children in the county. It is counter-intuitive that the catchment area of School A is more accessible than School B.\n\nFurthermore, the urban-rural dichotomy regarding quality of life caused by the Chinese dual system has been a long-standing issue (Chan and Wei, 2019; Ma et al., 2020). In the dichotomy, population and land management systems are both separate and unequal between urban and rural areas. The socialist governance system leads to the concentration of public resources in the urban areas, while rural areas lack the same resources. Xiao et al. (2017) identified that high access to urban parks occurred mainly in downtown Shanghai. Zhao and Cao (2020) investigated 81 million trips that used transit smart cards to identify the gradual decrease of job accessibility from the city centre towards the outer suburbs. In the case of hospitals, the urban-rural dichotomy of medical resources has already been identified, revealing a lack of medical resources in suburbs (Meng et al., 2015). In 2009, the Shanghai Municipal Government launched the ‘5+3+1’ project to promote the construction of high-quality hospitals in the periphery. Using this ‘a priori knowledge’, it seems impossible that the most abundant resources are located in the agricultural island in all three categories. Therefore, the 3SFCA model with non-weighted ratios might be the most appropriate method to reflect the accessibility distribution in Shanghai.\n\nIn summary, the reuse of weights, including the M2SFCA method (Figure 3 b-maps) and defining accessibility as the sum of weighted ratios (Figure 4), can generate an epistemological trap. The outcomes of access ratios can produce pattern stability incongruous with model features and resource types, when compared to the access results. Moreover, the side effect of reusing weights, which exaggerates accessibility for units with closer proximities and fewer suppliers, can produce counter-intuitive results. The reuse of weights may lead to methodologically plausible, but common-sense-defying distributions of accessibility. It is therefore necessary to combine empiricism and rationalism in the evaluation of accessibility methods; similarly, the use of geographic weights needs to be cautious and epistemologically consistent.\n\n\nDiscussion and conclusions\n\nSpatial equity and distributive justice have been controversial issues due to their elusive concepts and complex assessments (Guagliardo, 2004; Hay, 1995). Identifying spatial inequity requires recognising the areas with shortages of social resources. Accessibility, as a fundamental indicator of spatial equity, assesses the distribution of urban resources by integrating supply-demand ratios and distance decay into FCA metrics. However, varying parameters and flexible applications of FCA metrics could generate methodologically logistical but empirically counter-intuitive results.\n\nThis study reveals that the reuse of weights might lead to an epistemological trap and misperceptions of resource distribution. The reuse of weights in FCA metrics, which is the multiple use of weights in simulating the decay of supply capacity, population demand, and the supply-demand ratios, may exaggerate accessibility in marginal communities that are within fewer suppliers’ catchments. Whereas past researchers have found that catchment size should be set with caution (McGrail and Humphreys, 2009), the present study has shown the use of distance weights may also require vigilance. Comparing the results of access (the sum of supply-demand ratios) and access ratios (the sum of weighted supply-demand ratios) across three different resources shows that weighted ratios produce pattern stability beyond the model features of the E2SFCA, M2SFCA, and 3SFCA methods. On the contrary, non-weighted ratios may help to visualize the model characteristics most accurately. Linking the model results with empirical judgements, this study suggests that the 3SFCA method using non-weighted ratios can achieve the most realistic accessibility distribution among three resources in Shanghai.\n\nThere are at least three potential limitations concerning the results of this study. First, the results are limited by the city-level spatial scale and the topographic features of Shanghai. Smaller spatial scale and less complex environments could alleviate the exaggeration of weighted ratios. Shanghai’s polycentric city structure, as well as its islands, might also contribute to the problematic dispersed resource distributions. Further, the assessment is based on the local-level POI data and contains numerous supplier points, which can cause methodological fallacies in this specific combination of resource distribution, city structure, and unit aggregation. Fewer suppliers, even with divergent catchment areas, still generate simple topologies and may not encounter the trap of weights (Pan et al., 2018). Lastly, this study still produces place-based assessments with the FCA metrics, which lacks detail of the individual differences in facility utilization. Further studies can explore the solutions for identifying individual preferences in facility utilization and dealing with personal uncertainties.\n\nDespite these limitations, this study provides insights for a better understanding of accessibility and GIS modelling. For accessibility measurements, this examination of reused weights in FCA metrics raises the issue of evaluation criteria for improving methods. The socioeconomic attributes of accessibility measurement include social and cultural factors in the use of facilities and the organisation of resources. This socioeconomic attribute requires assessing improvements that have not only a methodologically logical necessity, but also a practical implication. This study reveals that sensible logic for the application of distance weights needs further exploration. Since the first law of geography argues that near things are more related than distant things (Tobler, 1970), the applications of distance weights have been a crucial method to represent this relationship. The study will prove useful in expanding our understanding of how distance weights should be applied in GIS models. Last but not least, the present study distinguishes the gap between methodological rational and empirical judgements in GIS modelling. It is difficult to discern whether the results of GIS models generated by rational techniques reflect the real situation. Understanding this reality is a matter of epistemology. Methodological fallacies, such as the trap of weights, need to be examined with empiricism and rationalism in further studies.\n\n\nData availability\n\nThe underlying data consists of the cell phone signalling data, road networks, point of interest (POI) data (primary schools, companies, and hospitals) in Shanghai in 2017, which was purchased from Urban Data Party (UDP) via its data service. These datasets cannot be made publicly available because they are under copyright to Urban Data Party. The following URLs are only available with Urban Data Party member registration:\n\n• Points of education facilities: https://www.udparty.com/index.php/detail/articledetails/?id=4502…title=19\\%E5\\%B9\\%B4\\%E4\\%B8\\%8A\\%E6\\%B5\\%B7\\%E5\\%B8\\%82\\%E6\\%95\\%99\\%E8\\%82\\%B2\\%E5\\%A4\\%A7\\%E7\\%B1\\%BBPOI\\%E6\\%95\\%B0\\%E6\\%8D\\%AE\n\n• Points of companies and hospitals: https://www.udparty.com/index.php/detail/articledetails/?id=1585…title=\\%E4\\%B8\\%8A\\%E6\\%B5\\%B7\\%E5\\%90\\%84\\%E7\\%B1\\%BBPOI\\%E6\\%95\\%B0\\%E6\\%8D\\%AE\\%E6\\%B1\\%87\\%E6\\%80\\%BB\n\n• Shanghai road network: https://www.udparty.com/index.php/detail/articledetails/?id=3820…title=\\%E4\\%B8\\%8A\\%E6\\%B5\\%B7\\%E5\\%B8\\%82\\%E9\\%81\\%93\\%E8\\%B7\\%AF\\%E6\\%95\\%B0\\%E6\\%8D\\%AE\\%EF\\%BC\\%882018\\%E5\\%B9\\%B411\\%E6\\%9C\\%88\\%EF\\%BC\\%89\n\n• Population density: Urban Data Party has since deleted the cell phone signalling data and updated the population density from WorldPop: https://dx.doi.org/10.5258/SOTON/WP00675.\n\nAs an alternative to the restricted UDP datasets (points 1-3), OpenStreetMap provides publicly available POI and road network data for Shanghai which is representative of the analysed datasets: https://www.openstreetmap.org/relation/913067. This data can be downloaded directly on the website via the Overpass API. The QGIS plug-in QuickOSM can also download the OpenStreetMap data and convert it to shapefiles, see tutorials here.\n\nZenodo: Python codes for accessibility and access ratios in the E2SFCA, M2SFCA, 3SFCA methods. http://doi.org/10.5281/zenodo.4890880 (Zhang, 2021).\n\nThis project contains the following extended data:\n\n• 01-E2SFCA.py\n\n• 02-M2SFCA.py\n\n• 03-3SFCA.py\n\n• LICENSE.txt\n\n• readme.txt\n\nData are available under the terms of the Apache License 2.0.\n\n\nAuthor endorsement\n\nProf. Dr. Frank Othengrafen confirms that the author has an appropriate level of expertise to conduct this research, and confirms that the submission is of an acceptable scientific standard. Prof. Dr. Othengrafen declares they have no competing interests. Affiliation: TU Dortmund University, Dortmund, 44149, Germany.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by China Scholarship Council (CSC) Grant number 201706260274.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe author would like to express sincere appreciation to René Westerholt at TU Dortmund University for his help and comments in the methodological discussion.\n\n\nReferences\n\nApparicio P, Abdelmajid M, Riva M, et al.: Comparing alternative approaches to measuring the geographical accessibility of urban health services: distance types and aggregation-error issues. Int J Health Geogr. 2008; 7: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArrow KJ: Rawl’s principle of just saving. Swedish J Economics. 1973; 75(4): 323–335. 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Publisher Full Text\n\nZhang X, Lu H, Holt JB: Modeling spatial accessibility to parks: a national study. Int J Health Geogr. 2011; 10(1): 31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao P, Cao Y: Commuting inequity and its determinants in Shanghai: New findings from big-data analytics. Transport Policy. 2020; 92: 20–37. Publisher Full Text" }	[ { "id": "99589", "date": "22 Nov 2021", "name": "Matthew R. McGrail", "expertise": [ "Reviewer Expertise Health services research - workforce distribution & access to health care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI congratulate the author for conducting this study. I agree that amongst the growth in use and testing of the broad family of FCA methods, it remains that catchment size and distance weights are under-researched. Some of my own research focused more so on catchment sizes; this study here focuses on the way that distance weights are applied.\nI’m confident that what the authors have tested and tried to present here is a significant addition to the evidence base. However, I’m still not 100% sure exactly what specifically the authors have actually tested – my expectation is that it will require minor clarification to address.\nThe authors specifically state early (in the Intro) that “for clarity, this study defines the original non-weighted accessibility as ‘access’ and the weighted ratios as ‘access ratios’. I have a high-level understanding the broad 2SFCA family of methods, but my confusion is tied to this statement (and thus how it is applied in the paper). Despite what Table 1 presents, I have not been able to understand what the Access (Ai) scores exactly relate to.\nIs non-weighted ‘access’ for the E2sfca equivalent to the original 2sfca (i.e. without weights), or something else?\n\nIn Table 1, the M2sfca Ai formula includes a weight, thus how is this non-weighted?\n\nI do not understand the difference between the Ai formula for E2sfca vs 3sfca? As per Figure 3, they clearly produce different answers but I can’ understand what specifically is being calculated differently – Table 1 suggests otherwise.\n\nClarification of specifically what the authors have done (e.g. relate then back to the equations (1) to (4)) will help enormously.\nSimilarly, am I to assume that the right column of Table 1 equivalent to the original 3 methods (written in a ‘short-hand’ way (like Figure 1), or have they applied some other modification?\nAgain, clarification of this ‘simple’ point will help enormously.\n\nThis misunderstanding aside, I really like what they’ve done in Figures 3 and 4. The results are presented and explained very clearly (even if I haven’t quite understood the underlying methods being compared). I also like the comparisons of the 3 types of resources. This gives the reader a broader understanding of the outcomes seen in Figs 3 and 4 and how each method relates to the different characteristics as per Table 2.\nI did not find Figure 2 helpful, it was quite confusing trying to understand specifically what it is trying to demonstrate at that point (it did not help me interpret Table 1, which is critical to the paper).\nApart from clarifying data sources and their GIS processes, the Methods section gives little detail on the specific formulae being tested (simply pointing back to Table 1). This is the critical information missing in the paper. As an aside, it is highly unusual for the Methods section to point the reader BACK to the Intro / Background sections for the detail.\nJust before the Discussion & Conclusion section, I really like their statement of “the reuse of weights may lead to methodologically plausible but common-sense defying distribution of accessibility”; however as per above, I think the reader is currently lacking some specific technical information of the methods to 100% appreciate this study’s evidence.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7570", "date": "13 Dec 2021", "name": "Lina Zhang", "role": "Author Response", "response": "The author would like to thank the reviewer for his kind comments and valuable questions. I have carefully read the referee's report and will revise the manuscript according to the referees' suggestions after the second reviewer’s report. I also feel quite sorry for the unclear articulation of the key concept in this first vision. It seems to be quite necessary to answer some questions mentioned in the report in advance. “The authors specifically state early (in the Intro) that “for clarity, this study defines the original non-weighted accessibility as ‘access’ and the weighted ratios as ‘access ratios. I have a high-level understanding the broad 2SFCA family of methods, but my confusion is tied to this statement (and thus how it is applied in the paper).” Response: This statement should be revised in the Intro. While distance weight has various applications in FCA methods, this study focuses on its application in the final step of FCA methods. The difference between ‘weighted’ and ‘non-weight’ refers to the second step in the 2sfca and the third step in the 3sfca method, which calculates the accessibility of one community to facilities. If we focus on the original 2SFCA method, then the first step is to calculate the supply-demand ratio of certain facility Rj and the second step is to calculate the sum of supply-demand ratio of certain unit Ai. For the second step, the very first method (2sfca) does not include distance weight () (Luo & Wang, 2003). However, there is another vision of this step in the enhanced-2sfca method, which multiplies the facility supply-demand ratio by distance weight () (Luo & Qi, 2009). The former one is the original non-weighted accessibility ‘access’, while the latter one is the ‘access ratio’. In the case of the 3sfca method, if we regard the first two steps (step 0: the calculation of supplier weight and step1: the calculation of Rj) as one finer step of Rj calculation, then the problem remains quite similar in the E2sfca method. The key question here is whether we should multiply Rj by distance weight and supplier weight in the final step ( vs. ). In the case of M2SFCA, I made a quite important mistake because of the complicated situation. And I believe this mistake raises major confusion. In Table 1, the M2sfca Ai formula is inaccurate. It should be . This mistake is made because I would emphasise the different calculation processes. In the previous two methods (E2sfca and 3sfca), the supply-demand ratio Rj belongs to one facility j. While in the M2sfca, the supply-demand ratio is calculated for each pair of facility j and population unit i. Because Delamater (2013) constructs a specific supply ratio for each facility j and unit i in the first step. To reflect this difference in the calculation process, I emphasized the wij in the first step of M2sfca in Table 1, which is unnecessary. However, the second step in the M2sfca is the same as the E2SFCA. So, the key question remains the same, that is, whether we should multiply Rj by the distance weight in the second step of the M2sfca. Therefore, the ‘access’ in M2sfca should be . And ‘access ratio’ should be . “Despite what Table 1 presents, I have not been able to understand what the Access (Ai) scores exactly relate to. Is non-weighted ‘access’ for the E2sfca equivalent to the original 2sfca (i.e. without weights), or something else? ” Response: No, the non-weighted ‘access’ for the E2sfca is not equivalent to the original 2sfca. Their difference lies in the first step, which is the calculation of supply-demand ratio Rj of certain facility j. In the original 2sfca, Rj is the supplier capacity divided by the population. In the E2sfca, Rj is the supplier capacity divided by the weighted population. The ‘non-weighted access’ for the E2sfca is the sum-up of . And the ‘weighted access ratio’ for the E2sfca is the sum-up of . “In Table 1, the M2sfca Ai formula includes a weight, thus how is this non-weighted?” Response: The M2sfca Ai formula is inaccurate. It should be The M2sfca Ai indeed includes distance weight, which is used to calculate the service supply ratio Rj in the first step. While this study focuses on the weight in the second step. The non-weighted ‘access’ in the second step refers to the access which sums up the Rij of each facility and each community without distance weight. On the contrary, the weighted ‘access ratio’ is to multiply each Rij by their distance weights wij. “I do not understand the difference between the Ai formula for E2sfca vs 3sfca? As per Figure 3, they clearly produce different answers but I can’ understand what specifically is being calculated differently – Table 1 suggests otherwise.” Response: I guess I should add one more paragraph to explain the difference between the Ai formula for E2sfca and 3sfca. The difference between the Ai formula for E2sfca and 3sfca lies in the calculation of facilities’ supply-demand ratio Rj. To be more precise, they differ mainly in the way they calculate the population demand. In E2sfca, the population demand of certain facility is the units’ population multiplied by distance weight. However, Wan et al. (2012) find this method is quite problematic. Because the 'unit' population is double-counted as the number of facilities increases. For example, if one unit has three facilities in its catchment, then the unit’s population is calculated three times. Each facility’s supply-demand ratio includes its population as demand. If the number of facilities in the unit’s catchment increases to ten, then this unit’s population is calculated ten times. This double-counted population causes enlarged demand in facility concentration areas and thus decreases the accessibility in those areas. To solve this problem, the 3sfca introduces one more step before the original Rj calculation, which divides the unit’s demand according to each pairwise weight of facility and unit. In 3sfca, the population demand of certain facility is the units’ population multiplied by distance weight and supplier weight. Similarly, am I to assume that the right column of Table 1 equivalent to the original 3 methods (written in a ‘short-hand’ way (like Figure 1), or have they applied some other modification? Response: Yes, the right column of Table 1 is equivalent to the original 3 methods. I guess this simple answer might remain some confusion on methodological understanding. So further details are provided as follow: If we divide the use of weights in FCA into two categories: one that applied in the calculation of facilities’ supply-demand ratio Rj, and the other applied in the calculation of accessibility, then the answer for this question might be clearer. The difference between non-weighted ‘access’ and weighted ‘access ratio’ is whether to multiply Rj by the distance weight, that is, the final step of FCA methods. The difference between methods (E2sfca, M2sfca, and 3sfca) is how to calculate Rj, that is, the first step of 2sfca methods and the first two steps of 3sfca. I hope the above explanation could clarify the key comparison (Ai vs. Ar) of this study. Further modifications to Figure 2 and the specific formulae in the Methods section's will be placed in the next version. References: Delamater, P. L. (2013). Spatial accessibility in suboptimally configured health care systems: a modified two-step floating catchment area (M2SFCA) metric. Health \\& Place, 24, 30-43. https://doi.org/10.1016/j.healthplace.2013.07.012 Luo, W., & Qi, Y. (2009). An enhanced two-step floating catchment area (E2SFCA) method for measuring spatial accessibility to primary care physicians. Health \\& Place, 15(4), 1100-1107. https://doi.org/10.1016/j.healthplace.2009.06.002 Luo, W., & Wang, F. (2003). Measures of spatial accessibility to health care in a GIS environment: synthesis and a case study in the Chicago Region. Environment and Planning B: Planning and Design, 30(6), 865-884. https://doi.org/10.1068/b29120 Wan, N., Zou, B., & Sternberg, T. (2012). A three-step floating catchment area method for analyzing spatial access to health services. International Journal of Geographical Information Science, 26(6), 1073-1089. https://doi.org/10.1080/13658816.2011.624987" }, { "c_id": "7691", "date": "08 Feb 2022", "name": "Lina Zhang", "role": "Author Response", "response": "This response is an additional explanation of the reviewer's report and provides further details of the second vision of the manuscript. First of all, the author would like to appreciate the referee for his thoughtful remarks and valuable suggestions. I revised the manuscript according to the referee’s suggestion. Some problems left in the previous response are answered as follows. The definitions of ‘access’ and ‘access ratios’ are not clear, including the statement in the Introduction and the formulas in Table 1.“The authors specifically state early (in the Intro) that “for clarity, this study defines the original non-weighted accessibility as ‘access’ and the weighted ratios as ‘access ratios’. I have a high-level understanding of the broad 2SFCA family of methods, but my confusion is tied to this statement (and thus how it is applied in the paper). Despite what Table 1 presents, I have not been able to understand what the Access (Ai) scores exactly relate to. ” Response: This statement in the Introduction has been revised. A detailed explanation has been added to the penultimate paragraph. And the difference between ‘weighted’ and ‘non-weight’, i.e. whether distance weights are applied in the last step of the FCA methods, has been highlighted in the first sentence of the last paragraph in the Introduction. The formulas and footnotes in Table 1 have been corrected and revised. The M2sfca Ai formula in Table 1 was inaccurate, which should not include weight. The explanation of the difference between the Ai formula for E2sfca, M2sfca, and 3sfca follows the Table 1 illustration, as shown below. “Table 1 shows the comparison of access Ai and access ratio Ar of the three methods. The difference between 'access' and 'access ratio' is whether to multiply the suppliers' supply-demand ratios (Rj) by distance weights (wij) in the last step. The differences between E2SFCA, M2SFCA, and 3SFCA is the way to calculate the suppliers' supply-demand ratios (Rj). The E2SFCA calculates Rj as supplier capacity divided by the weighted population. The M2SFCA argues that the supplier capacity also decreases while distance increases and multiplies supplier capacity by the distance weight. The 3SFCA reveals the double-counted population demand and multiplies population demand by the distance weight and the supplier weight.” Figure 2 provides limited information on the difference between ‘access’ and ‘access ratios’. “I did not find Figure 2 helpful, it was quite confusing trying to understand specifically what it is trying to demonstrate at that point (it did not help me interpret Table 1, which is critical to the paper).” Response: The equations of ‘access’ and ‘access ratio’ has been added in Figure 2. I also added a red dotted box to clarify the focus of Figure 2. In the same distribution of supplier and census grid, one grid’s access might be greater than the other, while whose access ratio is less than the other’s access ratios. It shows that different calculations of accessibility (the ‘access’ and ‘access ratio’ in this case) might lead to magnificent changes in accessibility distributions. “Apart from clarifying data sources and their GIS processes, the Methods section gives little detail on the specific formulae being tested (simply pointing back to Table 1). This is the critical information missing in the paper.” Response: I added the specific formulae being tested in the Study Design section of the Methods section. Besides, necessary parameters settings of the three FCA methods and three objects have also been added." } ] } ]	1	https://f1000research.com/articles/10-751
https://f1000research.com/articles/9-1252/v1	16 Oct 20	{ "type": "Research Article", "title": "Four practice-based preliminary studies on Human Givens Rewind treatment for posttraumatic stress in Great Britain", "authors": [ "Shona Adams", "Steven Allan", "William Andrews", "Keith Guy", "Jayne Timmins", "Elizabeth Barr", "Steven Allan", "William Andrews", "Keith Guy", "Jayne Timmins", "Elizabeth Barr" ], "abstract": "Background: Human Givens (HG) Rewind is a relatively unknown trauma-focussed treatment. This paper aimed to provide preliminary evidence of the effectiveness of Rewind to treat posttraumatic stress (PTS) in a variety of clinical settings in Great Britain. Methods: An observational prospective design was used in each of the four studies. Standardised questionnaires were administered in every session. Pre- and post-treatment means and effect sizes were calculated for each study, as were ‘recovery rates’ and ‘reliable change’ rates. Results: Across four studies, a total of 274 clients completed treatment and had complete data. The data capture rate ranged from 80-100%. The mean pre-treatment scores were in the severe range. The pre-post treatment effects sizes ranged from 1.90-2.68. The recovery rate, or percentage of clients who were below the clinical cut-off after treatment, ranged from 46-56% for the more conservative lower cut-offs, and ranged between 71-82% for the higher clinical cut-offs as used by Improving Access to Psychological Therapies (IAPT). Across the four studies, 83-96% of clients had ‘reliably improved’ (88-94% on trauma-specific questionnaires), with 4-17% having no reliable change on those questionnaires. There was no ‘reliable deterioration’. The mean number of HG treatment sessions ranged from 5-6.5 sessions (range 1–24 sessions), with between 73% and 84% of participants completing treatment in six sessions or less. Conclusions: These preliminary results indicate that HG Rewind appears to be a promising trauma treatment in a variety of settings. A randomised controlled trial is now required to determine the efficacy of this treatment.", "keywords": [ "Human Givens", "Rewind", "exposure", "imaginal exposure", "PTSD", "posttraumatic stress", "practice-based", "reliable change" ], "content": "Introduction\n\nPost traumatic stress (PTS) refers to clinical symptoms resulting from trauma and includes both symptoms and traumatic events that may not meet diagnostic criteria for posttraumatic stress disorder (PTSD). Traumatic events can result in PTSD but also depression, anxiety disorders, and substance misuse disorder (Brady et al., 2000; Schmidt, 2015). Survivors of significant traumas who do not meet all the DSM IV criteria for PTSD (American Psychiatric Association [APA]; 1994) can still suffer clinically significant impairment (Cukor et al., 2010) and can respond well to PTSD treatment (Dickstein et al., 2013; Handley et al., 2009). In addition, events that did not meet the DSM IV criteria of PTSD could be the content of intrusive images (Brewin et al., 2010; Day et al., 2004) and generate PTSD-like symptoms (Mol et al., 2005). This paper presents preliminary evidence for the effectiveness of a trauma-focused treatment called Rewind to treat PTS and PTSD.\n\nHuman Givens (HG) Rewind is a little known trauma treatment that was developed in the United Kingdom. It is a trauma-focused technique that utilises graded imaginal exposure in which the levels of psychological distancing from the trauma are graded during the exposure (Adams & Allan, 2019a). After explaining the technique and ensuring the trauma memory has been activated, the client is deeply relaxed and then ‘observes’ themselves watching an imaginary video of the traumatic event from before the event started to when the event is over, then ‘watches’ the video of the traumatic event, and finally experiences the traumatic event very quickly backwards (which is where “Rewind” gets its name)1. HG Rewind is part of HG therapy and was adapted by Griffin & Tyrrell (2004) from a technique called Visual Kinesthetic Dissociation (VKD; Bandler & Grinder 1979). The HG version of Rewind differs from the Muss version of Rewind that was also developed from VKD in Great Britain (Adams & Allan, 2018; Muss, 2002), in that the HG protocol includes steps to keep arousal levels relatively low during exposure. Reconsolidation of Traumatic Memories (RTM) is another trauma treatment technique that is derived from VKD (Gray & Bourke, 2015; Gray et al., 2019; Tylee et al., 2017).\n\nSeveral possible mechanisms explaining Rewind have been proposed (see Adams & Allan, 2018; Adams & Allan, 2019a; Adams & Allan, 2019b). Memory reconsolidation and extinction were suggested as mechanisms underlying VKD (Gray, 2010; Gray & Liotta, 2012). In memory reconsolidation the traumatic memory is retrieved and modified with updated non-fearful information (Schiller et al., 2010), and in extinction the prefrontal cortex inhibits the amygdala (Schiller et al., 2013) and the fear memory is only masked. Griffin & Tyrrell (2004) acknowledge the importance of REM sleep, the observing self, but proposed that that reduced arousal was the critical factor in processing trauma memories. Reduced arousal during exposure may keep the arousal within the ‘window of tolerance’. Adams & Allan (2019b) noted that psychological distancing is graded during exposure in Rewind. Psychological distancing involves observing the trauma from the perspective of an observer (Koenigsberg et al., 2010) rather than re-experiencing the trauma memory, and psychological distancing has been shown to be an effective method for emotional regulation (Ochsner & Gross, 2008) and has been used in other trauma treatments (e.g. Ehlers & Clark, 2000; Sloan et al., 2012; Sloan et al., 2013). Adams & Allan (2019a) summarised several mechanisms that might be involved in the reprocessing of the traumatic memory in Rewind, including the contextualisation of the traumatic event, activating the ‘observing self’ or ‘distancing’ (Griffin, 2005; Okhai, 2005), and that experiencing the trauma backwards during the ‘rewind’ stage may utilise a similar EMDR mechanism of ‘split attention’ (Lee et al., 2006; Lee et al., 2017) or the ‘orienting and relaxation response hypothesis’ (Pagani et al., 2017). For more details regarding the physiological mechanisms underlying memory consolidation, sleep, and connectivity between the amygdala and the prefrontal cortex, see Feng et al. (2018) and Murkar et al. (2018) and the link between the orienting response and REM sleep in processing memories see Pagani et al. (2017).\n\nRewind differs from other trauma techniques in that multiple traumas can be treated in one session (Guy & Guy, 2009; Murphy, 2007) making it potentially cost effective. As Rewind utilises imaginal exposure, the trauma does not need to be discussed in detail during treatment and preliminary evidence suggests that this may make treatment more accessible for people with shame-based traumas (Adams, 2017, pp. 136–137, 141). Potential increased treatment accessibility for some people and possible cost effectiveness suggest that Rewind is worthy of further investigation.\n\nHG Rewind is one of the techniques used in HG therapy. HG therapy is a brief solution-focused therapy that is based on a biopsychosocial model in which nine unmet basic emotional needs create heightened arousal to enable us to meet our needs (Griffin & Tyrrell, 2004). They proposed that we have natural resources to aid us that are adaptive but can also cause difficulties, such as the activation of the fight and flight response when we are not in danger. HG therapy integrates effective components of other therapies, such as anxiety management strategies, goal setting, skills training, use of imagery and imagery rescripting, metaphors and therapeutic use of language, graded exposure, and informally challenging negative thoughts, but within a different theoretical framework. Interventions to support fulfilling unmet emotional needs can include goal setting, social interventions like debt advice or housing provision, social skills training to facilitate socialising, goal setting to assist attaining a sense of status, finding a new job, and integrating into the community. Although not the main focus of treatment, some of these are recognised as part of a treatment programmes for those recovering from PTSD or PTS (e.g., Kintzle et al., 2018; Koven, 2018; Murphy, 2016), and HG therapy provides a psychological framework and rationale for these interventions. See Yates & Atkinson (2011) and Adams & Allan (2019a) for a more detailed description of basic HG techniques.\n\nAt present, the evidence for the effectiveness of Rewind is based on practice-based evidence. A literature review by Corps et al. (2008) only found individual case descriptions and two wider studies. The first of these studies by Guy & Guy (2003) reported on all those treated with Rewind during a specified period (N=30), with 40% rating the treatment as ‘extremely successful’, 53% rating it as ‘successful’, 7% rating it as acceptable, and no one rating the treatment as ‘poor’ or ‘a failure’. However, they did not use standardised questionnaires. The second of these studies by Murphy (2007) used a qualitative methodology to explore the effectiveness of a single Rewind treatment session. All those treated with Rewind at a trauma clinic were included (N=47). He reported that all those treated experienced symptom reduction with none meeting the criteria for PTSD after treatment, whether treated for single or multiple traumas. He also reported the Rewind could be used successfully as a standalone treatment or in conjunction with other treatments. Results from a more recent qualitative study using a single Rewind treatment session suggested that Rewind treatment may make treatment more accessible for shame-based traumas as details of the traumas do not need to be discussed during the treatment (Adams, 2017, pp. 136–141). A later uncontrolled study by Guy & Guy (2009) reported on 97 clients treated with the single HG Rewind treatment session. Mean scores on the Impact of Events Scale – Extended version (IES-E; Tehrani et al., 2002) reduced from 68 pre-treatment (the severe range) to 18 post-treatment (the normal range). However, only the pre- and post-treatment data were provided with no indication of effect sizes or standard deviations, making it difficult to compare their findings with other studies. Burdett & Greenberg (2019) reported 52% (N=504) of war veterans with planned endings treated with Human Givens therapy (42% of the whole sample) were below the clinical cut-off of 11 on Clinical Outcomes in Routine Evaluation (CORE-10; Connell & Barkham, 2007) after treatment. Of these, 42% attended more that one session and 5% attended 6 or more sessions. In a separate study comparing a single Rewind session with a treatment-as-usual session, data capture rates, means and effect sizes, as well as ‘reliable improvement’ and ‘recovery rates’ were reported (Adams & Allan, 2019b). After a single Rewind session (N=44), 38% were below the clinical cut-off of 11 on CORE-10, with a pre-post effect size of 1.86.\n\nThree studies have explored the effectiveness of HG therapy overall, have been reported (Andrews et al., 2011; Andrews et al., 2013; Tsaroucha et al., 2012). None of these three studies reported how many clients were treated with the Rewind technique. The first two of these studies were large trans diagnostic practice based studies that reported effect sizes as well as ‘recovery rates’ and ‘reliable improvement rates’ in line with the IAPT protocol (Clark et al., 2009). The Andrews et al. (2011) practice-based study included 3 therapists and 124 clients while the Andrews et al. (2013) study included 46 therapists at multiple sites, with 3,885 clients. Treatment was completed in an average of 3.75 treatment sessions (Andrews et al., 2011) and 4.69 treatment sessions (Andrews et al., 2013), excluding the initial assessment session. The pre-post treatment effect sizes were 1.39 (Andrews et al., 2011) and 1.58 (Andrews et al., 2013), both slightly above the IAPT effect size of 1.22 (Clark et al., 2009). Tsaroucha et al. (2012) compared HG treatment for depression with a treatment-as-usual control group in a non-randomised study and reported similar improvement rates between treatment groups but HG therapy requiring an average of 2 rather than 4 treatment sessions to achieve similar results. Thus, there is some preliminary evidence that HG therapy may be a promising treatment but it is unclear how frequently the Rewind technique was utilised in these studies.\n\nIn summary, Rewind is a relatively new treatment for PTS in which the trauma does not need to be discussed in detail and multiple traumas can be treated in one session. HG therapy that incorporates the Rewind technique may require fewer sessions and therefore might be cost effective, but more research is needed to record the number of treatment sessions for PTS. Practice-based evidence across different treatment settings has evolved to report ‘recovery rates’ and ‘reliable improvement’ as well as pre-post treatment effect sizes, and practice-based data capture rates have improved by using questionnaires in every session (see Clark et al., 2009). However, this data has not been consistently reported in previous Rewind studies and those earlier findings have yet to be replicated in different treatment settings.\n\nThis paper briefly describes the evidence from four practice-based preliminary studies for HG Rewind. The aim of this paper was to attempt to replicate findings from previous preliminary studies in a variety of settings. The average number of treatment sessions, exclusion criteria, data capture rates, effect sizes, recovery rates, and reliable improvement and deterioration are reported for each study.\n\n\nMethods\n\nEach of the four studies were observational prospective cohort studies. A session-by-session monitoring system was used where clients were asked to complete the questionnaires in every session in order to provide greater data capture rates (Clark et al., 2009; Gillespie et al., 2002). The scores from the last treatment session were used as the ‘end of treatment’ score.\n\nMeans, standard deviations and effect size were reported for each study. Cohen’s d was used to calculate the effect size, subtracting the post-treatment mean from the pre-treatment mean and dividing the outcome by the pooled standard deviation (Clark et al., 2009) using Microsoft Excel. Effect sizes were interpreted using Cohen’s (1988) suggested convention of 0.2 indicating a small, 0.5 a medium, and 0.8 a large effect size.\n\nA within-subject analysis was used to calculate ‘recovery rates’, ‘reliable improvement rates’, ‘no reliable change rates, and ‘reliable deterioration rates’. These measures of effectiveness (IAPT, 2012) have become more popular as IAPT has become established in Great Britain. ‘Recovery’ was defined as scores below the clinical cut-off on standardised questionnaires at the end of treatment. Participants were categorised as having ‘reliable improvement’, ‘no reliable change’, or ‘reliable deterioration’ using the Reliable Change Index (RCI) for each questionnaire. The RCI refers to the amount of change that is not likely to be due to chance. The RCI was calculated for those scales where the RCI was unknown using the Jacobson & Truax (1991) formula.\n\nThe Rewind technique involves graded imaginal exposure to the trauma(s), while keeping arousal levels relatively low. The Griffin & Tyrrell (2001) protocol was used. For a more detailed description and discussion of HG Rewind, see Adams & Allan (2019a). HG Rewind has several stages:\n\n1. Preparation and activation of the trauma memory. The therapist briefly explains the procedure and helps the client create an imaginary ‘video’ that will be used later in the process. The video starts with a good memory. Then for each trauma or trigger, the video starts before and ends when the incident is over, and then the video ends with a recent good memory.\n\n2. Relaxation. The therapist guides the client to become deeply relaxed. This includes imagining being in a relaxed place.\n\n3. Double distancing exposure. In their imagination, the client watches themselves watch the imaginary ‘video’ backwards and forwards until they feel calm when watching themselves.\n\n4. Single distancing exposure and association exposure. The client watches the imaginary video with the trauma memories backwards and forwards until they are calm when watching it. For the association exposure, the client is then guided to re-experience all of the trauma sensations very quickly backwards by imagining they are being pulled backwards very quickly through the memory from when the trauma was over to the beginning before the trauma started. This is repeated until the scenes evoke no anxiety.\n\n5. Rehearsal (optional). If relevant, the client is asked to visualize themself reacting to a similar situation in the future in a way that the client would like to respond.\n\n6. End. The client is re-oriented to the present.\n\nThe procedures and measures used in each study are described below. Data collected at each of the sites was part of the routine clinical care, and as such did not require ethical approval from the University of Leicester. However, all participants provided informed consent for their anonymised data to be used to study the effectiveness of their treatment.\n\nThe purpose of this preliminary study was to investigate the effectiveness of the Rewind treatment for PTS in a police force setting. Officers and staff in the police force were referred for treatment by the Occupational Health advisors, and all those who were referred were treated with no exclusion criteria. Any staff with a possible diagnosis of PTSD were first be seen by the Forces Medical Advisor for a diagnosis before making the referral for Rewind treatment. All the clients treated using the Rewind technique in a specific police force between 2009 and 2013 were included. All those treated by the service completed the CORE-OM before and after treatment. Those with a diagnosis of PTSD or PTS were also given the IES-R before and after treatment. The Griffin & Tyrrell (2004) HG treatment was used with the Griffin & Tyrrell (2001) protocol for the Rewind technique.\n\nImpact of Events Scale – Revised version (IES-R). The IES-R is a modified version of the IES (Horowitz et al., 1979), and is a questionnaire designed to measure symptoms of traumatic stress. The IES has an avoidance and intrusion subscale. Weiss & Marmar (1997) added an intrusion subscale with seven additional items as part of the IES-R to conform with DSM IV (APA, 1994) PTSD criteria. There are ‘avoidance’, ‘hyper-arousal’, and ‘intrusion’ subscales that are added together to give a total score. In the IES-R, the respondent answers the question in relation to the impact of a specific traumatic event on symptoms using a 0–4 rating scale. The IES-R has 22 items and can generate a total score of 88. A clinical cut-off of 33 has been established (Creamer et al., 2003). The RCI for the IES-R is 9 (IAPT, 2011).\n\nClinical Outcomes for Routine Evaluation (CORE-OM). The CORE-OM was designed in the UK by Evans et al. (2000) to measure outcomes for psychological therapies. It is a 34-item questionnaire, where items are scored from 0–4 over the past week, and cover subjective well-being, symptoms, functioning, and risk. It has good internal consistency and test-retest reliability (Evans et al., 2002). Scores of 8.5–12.5 are mild, 12.75 – 16.75 are moderate, 17–21 are moderate to severe, with scores over 21 considered to be severe. An RCI on the CORE-OM is 5 or more (Connell & Barkham, 2007). The recommended cut-off between clinical and non-clinical populations for the CORE-OM is 10 (Connell et al., 2007).\n\nThe purpose of this preliminary study was to investigate the effectiveness of the Rewind treatment for war veterans suffering from PTS in clinical settings using 43 therapists. All war veterans who self-referred between 24 April, 2014 and 14 January, 2015 for treatment to a national war veteran charity providing HG therapy were included. The charity then referred the veterans to fully qualified and registered local HG therapists. The charity registered clients online on the Pragmatic Research Tracker when the referral was made, ensuring all those referred to the service were eligible to be included. HG therapy included the Griffin & Tyrrell (2001) Rewind treatment protocol for trauma. All the therapists were trained HG therapists and on the HG national register.\n\nImpact of Events Scale –Extended Version (IES-E). The IES-E is a modified version of the Impact of Events Scale (IES; Horowitz et al., 1979). Tehrani et al. (2002) modified the IES by adding a question about sleep to the ‘intrusion’ subscale and adding a ‘hyper-arousal’ subscale to make it more in line with the DSM IV (APA, 1994) PTSD criterion. The respondent answers the questions in relation to the impact of a specific traumatic event on symptoms on a 0–4 rating scale. There are ‘avoidance’, ‘hyper-arousal’, and ‘intrusion’ subscales which are added together to give a total score. There are 23 items and the maximum score is 92. Using a UK sample, Tehrani et al. (2002) found the reliability and discriminant validity of the IES-E to be good. The mean Cronbach’s alpha for the three subscales was 0.92, and the standard deviation from the norm was 12.6. Using the Jacobson & Truax (1991) formula, the RCI for the IES-E is 6. Tehrani et al. (2010) determined the clinical cut-off for PTSD for the IES-E was 50 and above, with scores of 40–49 indicating moderate symptoms still requiring treatment, scores of 30–39 indicating mild symptoms, and scores of less than 30 being in the normal range.\n\nClinical Outcomes in Routine Evaluation (CORE-10). The CORE-10 (Connell & Barkham, 2007) is a brief 10 item standardised questionnaire measuring psychological distress that is based on the longer 34 item CORE-OM (Barkham et al., 2001). Each self-report item is rated on a 0–4 scale, with a total score of 40. CORE-10 has a high correlation with CORE-OM (Adams, 2017; Andrews et al., 2011; Andrews et al., 2013). The clinical cut-off for CORE-10 is 11, with the specific cut-off for depression of 13 (Barkham et al., 2013; IAPT, 2011). The RCI is 6 or more (Barkham et al., 2013).\n\nThe purpose of this preliminary study was to examine the clinical effectiveness of a single Rewind session to treat PTS in a clinical setting. All clients who were referred to the PTSD treatment clinic between 2009 and 2014 were included and accepted for treatment with no exclusion criteria. The Griffin & Tyrrell (2001) Rewind protocol for treating trauma was used. This treatment session was part of the HG therapy that they received. The Impact of Events Scale –Extended Version (IES-E) was administered to clients before the Rewind treatment session and in the follow-up session, 1–2 weeks after the treatment.\n\nImpact of Events Scale –Extended Version (IES-E). See above.\n\nThe purpose was to evaluate the effectiveness of HG therapy for treating PTS in a fourth clinical setting. All clients who were referred to this private practice were treated with Rewind between February 2010 and February 2015 were eligible for inclusion in this study. Of the 113 clients treated during this period, 38 were treated with HG therapy without the Rewind and 75 were treated with HG therapy including Rewind. There were no exclusion criteria, with all those being referred for treatment being accepted for an assessment. The CORE-10 (study 4a) and the GAD-7 and PHQ-9 (study 4b) were administered at the assessment and at every subsequent treatment session. The questionnaires used in this practice changed during the data collection period to reflect changes in practice in the local area. The Griffin & Tyrrell (2001) Rewind protocol was used.\n\nClinical Outcomes in Routine Evaluation (CORE-10). See above\n\nGeneralised Anxiety Disorder (GAD-7). The GAD-7 is a self-report questionnaire to screen and measure the severity of generalised anxiety disorder (Spitzer et al., 2006). It has 7 items that are rated on a 4 point scale. The scoring range on the GAD-7 is 0–21, with scores of 15–21 indicating severe anxiety, scores of 11–15 indicating moderate anxiety, and scores of 5–10 indicating mild levels of anxiety. The clinical cut-off for anxiety on the GAD-7 is 8 and above, and the RCI is 4 or more (Clark & Oates, 2014)\n\nPatient Health Questionnaire (PHQ-9). The PHQ-9 is a self-report questionnaire to screen and measure the severity of depression (Kroenke et al., 2001). It has 9 items that are rated on a 4 point scale, with scores ranging from 0–27. Scores of 20–27 indicate severe depression, scores of 15–19 indicate moderately severe depression, scores of 10–14 are in the moderate range, scores of 5–9 are in the mild range, with the clinical cut-off for depression being 10 and above. The RCI is 6 or more (Clark & Oates, 2014).\n\n\nResults\n\nThe anonymised data is available as Underlying data (Andrews, 2020; Barr, 2020; Guy, 2020; Timmins, 2020).\n\nDemographics are reported in Table 1. There were approximately 30% males in two of the studies and 87% male in the war veteran study. The gender of participants in one study was unknown. The average age in the studies ranged from 39 years to 46 years, with age in one study not recorded.\n\n†Note the gender of some participants was not recorded.\n\nThe client pathways for all studies, including those referred, assessed, started treatment, completed treatment and data capture rates for ‘intention-to treat’ and those who completed treatment for all studies are summarised in Table 2. Studies 1, 2, and 3 treated everyone that was assessed. Clients were excluded from these studies if they were under 18 years old (n=5) or were below the clinical cut-off before treatment commenced (n=15). The intention-to-treat data capture rates ranged from 80% to 100%. The data capture rates for those who completed treatment were 100% for three studies and 94% for Study 2.\n\n† One person died, two did not have cognitive capacity during the assessment.\n\n‡ Two were below the clinical cut-off prior to treatment.\n\n§ Thirteen were under the clinical cut-off prior to treatment, five wanted consultation about a family member not individual treatment, and five were under 16 years old.\n\nTable 1 shows the number of sessions in each study. The results for Study 3 are based on a single Rewind session, but participants had other HG therapy treatment sessions. The mean number of sessions in all the studies ranged from 5 to 6.5, with between 73% and 84% completing treatment in 6 sessions or less. The number of treatment sessions ranged from one to 24 sessions.\n\nResults for those who completed treatment in all of the studies are presented in Table 3. This includes the number of those who completed treatment, mean and standard deviations for pre- and post-treatment scores with the clinical cut-off for that questionnaire, the mean change in scores and the RCI for the questionnaire, and the effect size. The mean pre-treatment scores were either in the severe range or in the moderately-severe range on all questionnaires. Many of the questionnaires had both a higher and a lower clinical cut-off. The mean post-treatment scores were below the higher clinical cut-off in all of the studies and below the lower clinical cut-off in Studies 1 and 4b. The change in scores for all of the studies was higher than the RCI. There was a large pre-post treatment effect size for each of the studies.\n\n†= Primary questionnaire of study; ‡ = In the severe range for the questionnaire; § = Below clinical cut-off; ¶ = Below clinical higher cut-off but above lower cut-off.\n\nThe recovery rates, reliable improvement rates, no reliable improvement and reliable deterioration for participants who completed treatment are presented in Table 4. With the trauma questionnaires, the recovery rates ranged between 45% – 52% for the lower cut-offs (normal range) and 47%–89% for the higher clinical cut-offs. Using the CORE questionnaires the recovery rates ranged from 49% – 59% for the lower clinical cut-off and 58% – 74% for the higher IAPT clinical cut-off.\n\n† The Coffey et al. (2006) clinical cut-off of 27 for the IES has a sensitivity rate of 91% based on a sample of 99, whereas the clinical cut-off of 35 suggested by Neal et al. (1994) is used in many studies but was only based on a sample of 36.\n\n‡ Clinical cut-offs for depression (Barkham et al., 2013; IAPT, 2011; Kroenke et al., 2001).\n\n§ Scores of < 30 are within the normal range on the IES-E and scores of > 50 indicate probable PTSD (Tehrani et al., 2010).\n\nThe improvement rate of those who were either above the RCI or below the clinical cut-off after treatment ranged from 88% – 94% for trauma symptoms, and 83% – 96% on the other questionnaires. That is, between 9% –17% of the veterans and 4% – 13% of the other clinical groups in these studies had no ‘reliable improvement’. There was no ‘reliable deterioration’ in any of the studies.\n\n\nDiscussion\n\nThe aim of this paper was to report the results of four practice-based preliminary studies on HG Rewind in Great Britain. In these four studies, a total of 274 clients completed treatment by 46 therapists and had complete data, with a data capture rate of 80–100%. The mean pre-treatment scores were in the severe or moderately-severe range on all questionnaires. Mean number of treatment sessions ranged from 5–6.5, with 73–84% of treatment being completed in 6 sessions or less. The effect sizes ranged from 1.90–2.68, with the lowest effect sizes in the study with 43 therapists. The recovery rate, or percentage of clients who were below the clinical cut-off after treatment, ranged from 46–56% for the more conservative lower cut-offs, and ranged between 71–82% for the higher clinical cut-offs used in IAPT. Across the studies, 83–96% had ‘reliably improved’ (88–94% on trauma-specific questionnaires), with around 10% (4–17%) having no reliable change on those questionnaires. There was no ‘reliable deterioration’.\n\nThese results are similar to previous studies using HG therapy. The results of Study 3 are from a single Rewind treatment session, with 45% of the sample was below the clinical cut-off of 30 on the IES-E after the Rewind treatment. Other preliminary studies have found that some clients do not require further treatment after a single Rewind session. Of those treated with Rewind in a trauma clinic in Northern Ireland (N=44), 61% of those treated were reported as only needing a single Rewind session (Murphy, 2007). Similarly, of those treated with a single Rewind session at a pilot NHS trauma clinic (N=27), 37% did not need further treatment (Adams, 2017, p.133) and in a separate study (N=44), 38% were below the clinical cut-off of 11 on the CORE-10 after a single Rewind session (Adams & Allan, 2019b). Burdett & Greenberg (2019) reported 58% (N=504) only attended one session, although it is unclear how many of these did not require further treatment. With the emphasis on cost-effective treatment the possibility of a stepped care approach to trauma treatment might be considered, with a single trauma-focussed exposure treatment being offered initially to those who might benefit from it before being stepped up to currently recommended treatments if necessary.\n\nWhile a single treatment session can be effective, most clients require more than one HG treatment session. In spite of this, the mean number of HG treatment sessions in these four pilot studies ranged from 5–6.5 sessions (range 1–24 sessions). This is similar to other HG studies. Guy & Guy (2009) treated their clients in six sessions or less. Andrews et al. (2013) reported an average of 4.69 treatment sessions (N=3,885) and Andrews et al. (2011) reported an average of 3.75 treatment sessions (N=124). It is important to note that across the four studies presented here, 16–27% of these clients required more than six sessions. Burdett & Greenberg (2019) reported 5% (N=504) attended six or more sessions. Thus, although the majority of clients treated with HG therapy were treated within six treatment sessions, it is also clear that some clients require more sessions.\n\nThe pre-post treatment effect sizes across the four studies ranged from 1.90–2.68 for those who completed treatment. This is in line with other HG studies, where the pooled effect sizes for the IES-E were 2.13 (Guy & Guy, 2009) and for the CORE-10 were 1.86 (Adams & Allan, 2019b), 1.58 (Andrews et al., 2013), and 1.39 (Andrews et al., 2011).\n\nRecovery rates using CORE-10 in these studies ranged from 49–54%. Similarly, 56% (N=124) and 54% (N=3,885) were below the CORE-10 clinical cut-off of 11 after HG treatment (Andrews et al., 2011 and Andrews et al., 2013 respectively). For war veterans completing treatment, 52% (N=504) were below the CORE-10 clinical cutoff after treatment (Burdett & Greenberg, 2019). The IES-E clinical cut-off for PTSD is 50 and above, with scores less than 30 considered in the normal range. In Study 3, 80% were below the higher IES-E cut-off of 50 after treatment with 45% in the normal range. While these scores are not directly comparable, in Study 1, 47% were in the normal range with a score below 33 on the IES-R, and in Study 2, 52% were below the Coffey et al., (2006) IES clinical cut-off of 27 whereas 89% were below the higher IES clinical cut-off of 35 suggested by Neal et al. (1994). Nonetheless, all of these HG recovery rates exceed the UK Department of Health’s target for IAPT services of a 40% recovery rate (IAPT, 2012).\n\nIn summary, the results of these four studies seem to be in line with previous evidence for HG therapy, both in terms of outcomes and the number of sessions.\n\nWhile these studies had high data capture rates and low exclusion criteria, subjects were not randomized nor were control groups used. As such, no firm conclusions can be drawn about the effectiveness of the treatment. Data on ethnicity was not collected. Studies all relied on self-report measures. Referring parties made all PTSD diagnoses and were not part of the study protocol. In addition, not all of the participants had a PTSD diagnosis, and therefore no conclusions about PTSD treatment should be drawn from these results. There was no long-term follow-up.\n\n\nConclusion\n\nThe effect sizes and recovery rates in these uncontrolled studies suggest that HG Rewind is potentially an effective treatment for PTS. These studies appear to support previous research, which indicated that a single session of Rewind treatment could treat a significant number of clients. While some clients may need more sessions, the majority of clients were treated in six HG sessions or less. This suggests that HG Rewind may be a cost effective treatment, but more research is needed to evaluate this. Further research is also needed to determine the effectiveness of Rewind in other cultures. Finally, an RCT with long-term follow-up is now clearly needed.\n\n\nData availability\n\nFigshare: Timmins (2020) Study 1 data, Human Givens Rewind treatment for posttrauamtic stress in a police force, https://doi.org/10.6084/m9.figshare.11316041.v2 (Timmins, 2020).\n\nFigshare: Andrews (2020) Study 2, Human Givens Rewind treatment of posttrauamtic stress in veterans. https://doi.org/10.6084/m9.figshare.11316032.v3 (Andrews, 2020)\n\nFigshare: Guy (2020) Study 3 data, Human Givens Rewind treatment for posttraumatic stress and PTSD. https://doi.org/10.6084/m9.figshare.11316038.v2 (Guy, 2020).\n\nFigshare: Barr (2020) Study 4, Human Givens Rewind treatment for posttrauamtic stress. https://doi.org/10.6084/m9.figshare.11316035.v2 (Barr, 2020).\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\nNotes\n\n1 A more detailed description of the Rewind technique is provided later in this paper.", "appendix": "References\n\nAdams S: Human Givens Rewind treatment for PTSD and sub-threshold trauma. 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Reference Source\n\nHandley RV, Salkovskis PM, Scragg P, et al.: Clinically significant avoidance of public transport following the London bombings: Travel phobia or subthreshold posttraumatic stress disorder? J Anxiety Disord. 2009; 23(8): 1170–1176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorowitz M, Wilner N, Alvarez A: Impact of Events Scale: A measure of subjective stress. Psychosom Med. 1979; 41(3): 209–218. PubMed Abstract \| Publisher Full Text\n\nImproving Access to Psychological Therapies (IAPT) National Team: Enhancing Recovery Rates in IAPT Services: Lessons From Year One. London: National IAPT Programme Team. 2011.\n\nImproving Access to Psychological Therapies (IAPT): IAPT Key Performance Indicator (KPI) Technical Guidance for Adult IAPT Service 2012/13. 2012.\n\nJacobson NS, Truax P: Clinical significance: A statistical approach to defining meaningful change in psychotherapy research. J Consult Clin Psychol. 1991; 59(1): 12–19. PubMed Abstract \| Publisher Full Text\n\nKintzle S, Barr N, Corletto G, et al.: PTSD in U.S. Veterans: The role of social connectedness, combat experience and discharge. Healthcare (Basel). 2018; 6(3): 102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoenigsberg HW, Fan J, Ochsner KN, et al.: Neural correlates of using distancing to regulate emotional responses to social situations. Neuropsychologia. 2010; 48(6): 1813–1822. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoven SG: Veteran treatments: PTSD interventions. Healthcare. 2018; 6: 64.\n\nKroenke K, Spitzer RL, Williams JBW: The PHQ-9: Validity of a brief depression severity measure. J Gen Intern Med. 2001; 16(9): 606–613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee CW, Taylor G, Drummond PD: The active ingredient in EMDR: is it traditional exposure or dual focus of attention? Clinical Psychology and Psychotherapy. 2006; 13(2): 97–107. Publisher Full Text\n\nLee JLC, Nader K, Schiller D: An update on memory reconsolidation updating. Trends Cogn Sci. 2017; 21(7): 531–545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMol SSL, Arntz A, Metsemakers JFM, et al.: Symptoms of post- traumatic stress disorder after non-traumatic events: Evidence from an open population study. Br J Psychiatry. 2005; 186: 494–499. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurkar AA, De Koninck J: Consolidative mechanisms of emotional processing in REM sleep and PTSD. Sleep Med Rev. 2018; 41: 173–184. PubMed Abstract \| Publisher Full Text\n\nMurphy D: Supporting veterans. Therapy Healthcare Counselling and Psychotherapy Journal. 2016; 16(2): 24–27.\n\nMurphy M: Testing treatment for trauma: Qualitative research providing evidence of effectiveness of the rewind technique for trauma. Human Givens. 2007; 14(4): 37–42.\n\nMuss DC: The rewind technique in the treatment of post-traumatic stress disorder. In Figley, C. R. (Ed.), Brief treatments for the traumatized, a project of the Green Cross Foundation. Westport, CT: Greenwood. 2002; 306–314. Reference Source\n\nNeal LA, Walter B, Rollins J, et al.: Convergent Validity of Measures of Post-Traumatic Stress Disorder in a Mixed Military and Civilian Population. J Trauma Stress. 1994; 7(3): 447–455. PubMed Abstract \| Publisher Full Text\n\nOkhai F: Tapping versus rewind. Human Givens. 2005; 12(2): 45–46.\n\nOchsner KN, Gross JJ: Cognitive emotion regulation: Insights from social cognitive and affective neuroscience. Curr Dir Psychol Sci. 2008; 17(2): 153–158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPagani M, Amann BL, Landin-Romero R, et al.: Eye movement desensitization and reprocessing and slow wave sleep: A putative mechanism of action. Front Psychol. 2017; 8: 1935. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchiller D, Kanen JW, LeDoux JE, et al.: Extinction during reconsolidation of threat memory diminishes prefrontal cortex involvement. Proc Natl Acad Sci U S A. 2013; 110(50): 20040–20045. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchiller D, Monfils MH, Raio CM, et al.: Preventing the return of fear in humans using reconsolidation update mechanisms. Nature. 2010; 463(7277): 49–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmidt U: A plea for symptom-based research in psychiatry. Eur J Psychotraumatol. 2015; 6: 27660. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSloan DM, Lee DJ, Litwack SD, et al.: Written Exposure Therapy for Veterans Diagnosed with PTSD: A Pilot Study. J Trauma Stress. 2013; 26(6): 776–779. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSloan DM, Marx BP, Bovin MJ, et al.: Written Exposure as an Intervention for PTSD: A Randomized Clinical Trial with Motor Vehicle Accident Survivors. Behav Res Ther. 2012; 50(10): 627–635. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpitzer RL, Kroenke K, Williams JB, et al.: A Brief Measure for Assessing Generalized Anxiety Disorder: The GAD-7. Arch Intern Med. 2006; 166(10): 1092–1097. PubMed Abstract \| Publisher Full Text\n\nTehrani N, Cox SJ, Cox T: Assessing the impact of stressful incidents in organizations: The development of an extended impact of events scale. Couns Psychol Q. 2002; 15(2): 191–200. Publisher Full Text\n\nTehrani N, Rainbird C, Dunne B: Supporting the police following the 7/7 London terrorist bombs. In N. Tehrani (Ed.), Managing Trauma in the Workplace: Supporting workers and organisations. London: Routledge. 2010; 191–203.\n\nTsaroucha A, Kingston P, Stewart T, et al.: Assessing the effectiveness of the “human givens” approach in treating depression: A quasi experimental study in primary care. Mental Health Review Journal. 2012; 17(2): 90–103. Publisher Full Text\n\nTimmins J: Timmins (2020) Study 1 data, Human Givens Rewind treatment for posttrauamtic stress in a police force. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11316041.v2\n\nTylee DS, Gray R, Glatt SJ, et al.: Evaluation of the reconsolidation of traumatic memories protocol for the treatment of PTSD: a randomized, wait-list-controlled trial. J Mil Veteran Fam Health. 2017; 3(1): 21–33. Publisher Full Text\n\nWeiss DS, Marmar CR: The impact of event scale – revised. In J. Wilson & T. M. Keane (Eds.), Assessing Psychological Trauma and PTSD. New York: Guilford Press. 1997; 399–411.\n\nYates Y, Atkinson C: Using Human Givens therapy to support the well-being of adolescents: A case example. Pastoral Care in Education. 2011; 29(1): 35–50. Publisher Full Text" }	[ { "id": "73276", "date": "27 Oct 2020", "name": "Richard Gray", "expertise": [ "Reviewer Expertise I am the author of several of the articles cited here. I have done extensive research in the neuroscience of and behavioral interventions for PTSD", "and addiction. I have significant research experience in behaviorism", "hypnosis", "NLP", "and Jungian theory." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important paper that continues the process of documenting supporting evidence for the rewind technique in the Human Givens program of treatment as begun by Adams and subsequently reported in Adams and Allan, 2019.1 The intervention first appears in the Human Givens literature with Guy and Guy’s qualitative review of the intervention (Guy & Guy 2003).2 Every effort appears to have been made to report all of the relevant studies. The technique is adequately detailed here and elsewhere. In light of the preliminary nature of the reports, the statistical methods are adequate. The review is also important because it represents a growing body of emerging evidence for the use of NLP-based therapies as an element in mainline treatment protocols. The authors accurately source the technique to its historic roots in NLP and David Muss’ heroic (1991, 2002) work with veterans, service members and first responders.3,4 The authors review four studies that use the intervention to treat Post Traumatic Stress, and presumably, prodromal PTSD. Two versions of the Impact of Events Scale (IES) are used to provide a presumptive diagnosis of PTSD, and in all studies, any patient not scoring above the minimal diagnostic cut-off (32-33 depending upon the version used) was excluded. Similarly, the studies report using two different versions of the UK-based Clinical Outcomes for Routine Evaluation (CORE; CORE-OM, Evans, et al., 2002; CORE-10, Connell & Barkham, 2007) for the evaluation of overall function.5,6 The two versions of the inventory have clinical cutoff scores of 10 and 11 respectively.\n\nThe study reports reliable change indices for each measure. The intervention produced reliable symptom score reductions on both measures. This suggests that the intervention, like the mainline treatments for PTSD, produces reliable symptom reductions independent of total remission. Pre-post effect sizes for the four studies were almost uniformly above 2 SMDs.\n\nFollow-up times were only reported for study 3. It would be useful if the authors could provide an indication of follow-up intervals from the other studies. If longer-term follow-ups have been collected, they would provide some important data that could reveal whether the results are robust and may also provide information regarding the underlying mechanism. In this regard, without a clear sense of when the follow-up data was obtained and whether longer term assessments were made, the use of the term ‘recovery’ may be a bit ambitious. Similarly, the lack of reliable deterioration must, in light of the missing post treatment follow-ups, be interpreted as reliable deterioration in the course of treatment. Please clarify this. Although the current review is limited to practice-based studies without controls, they form an excellent base for effectiveness research in which the outcomes of a new treatment are compared against the results of more classical treatments (Begley, 2011; Cohen, 2013; Docteur & Berenson, 2010).7,8,9 When one considers that this process obtains the same general level of symptom score reductions--in the course of six treatments or fewer--it may be profitably compared to mainline treatments for PTSD used in the US and elsewhere. In this regard, I would strongly suggest a review of Steenkamp et al.’s report of psychological treatments for military PTSD (Steenkamp et al., 2015).10 Without referring to the citation, it is unclear whether the qualitative studies reported on page 2 of the study occur in the context of the broader Human Givens treatment program or not (they do). Please clarify this in the text. The text reporting the measures used in study 2 (page 5), indicates that the IES-E was used. That designation is missing in Tables 3 and 4. They should be amended. In the references, there is an anomalous “15” in the Bandler and Grinder Citation. Please correct this.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7415", "date": "07 Feb 2022", "name": "Shona Adams", "role": "Author Response", "response": "Many thanks for your very thoughtful review. Thank you for noting the two typographical errors in Tables 3 and 4 and the one in the References. The error in Table for describes the “IES” used in Study 2 instead of the “IES-E” which is described in the methods section for Study 2. A check was done with the data and confirmed that the “IES-E”. The clinical cut-offs for IES-E was corrected in Table 3 and the number below the correct clinical cut-off in Table 4 was recalculated and triple checked. These have been corrected. You are correct that unfortunately there were not follow-up intervals in three of the four studies. This information has been added to the Results section and in Table 2. This is indeed a weakness of these studies and is already mentioned in the Limitations section and also in future research recommendations in the Discussion. Regarding the terminology of “recovery rates”, I agree with your statement about not using the word “recovery” when relating to results that don’t have follow-up, because it is uncertain from the data if the positive results were maintained. We have changed reference to the reported “recovery rates” in our studies to “below cut-off rates”, “below the clinical cut-off” or “within the normal range after treatment”. You statement about reliable deterioration has been noted. While some participants had ‘no reliable change’, there was no ‘reliable deterioration’ in any of the studies. We modified the statement in the discussion to state “There was no reliable deterioration reported in any of the studies at the end of treatment.” The statement in the discussion about reliable deterioration has been removed as requested. Many thanks for your suggestion to reference Steenkamp et al. (2015) review of RCTs for military-related PTSD. While reluctant to provide direct comparisons, we have included this review as you suggested for context. We have added a section on veterans that includes this reference and some more recent studies and relating to this population and to Study 2 in this paper. While we are presenting this data, and as there are not head to head comparisons, no direct comparisons can be made about the different treatments, but seeing this data in the context of other data does put the results into context. I note the Steenkamp et al. (2020) call for “improvement in existing PTSD treatment and for development and testing of novel evidence-based treatments”, and this is what the present paper aims to do. Your comment on “comparative effectiveness” (CE) in practice-based literature in the three articles mentioned has been noted. Indeed, Docteur and Berentsein (2010) stated that comparative effectiveness can indicate treatments being more cost-effective compared to the standard treatment. Also noted was the suggestion that applying new research methods using databases “could replace traditional ‘hierarchies of evidence’ and reliance on experimental methods”. High quality practice-based methods, with high data capture rates, session by session data collection, intention to treat reporting, and low exclusion criteria can arguably be more reliable in determining effectiveness in clinical settings than poor quality randomised controlled trials (RCTs) with high attrition rates, low data capture rates and many exclusion criteria. In fact, it could be argued that instead of controlling for extraneous variables through randomisation, it is possible that ‘introducing variability’ which is possible in practice-based studies, such as multiple therapists at multiple sites with large numbers of participants could also control for the effect of extraneous variables. Then if the results at the different sites with different therapists using the same standardised measures are similar to those found in effective treatments in RCTs and greater than the control groups in those studies, one could argue that the improvements are due to the treatment in the practice-based studies rather than extraneous variables. However, while CE can refer to comparisons to benchmarks it often refers to head to head studies in real life populations or to data in databases and for this reason this term was not used in this paper. Many thanks again for your attention to detail and your thoughtful and helpful comments. An additional statement was also added to the Limitation section. According to the British Psychological Society (2019, p. 9), “psychologists should ensure that their language and practice does not inadvertently exclude GSRD [gender, sexuality and relationship diverse] people”. Following APA guidelines (APA, 2015; APA, 2020), this paper refers to ‘gender’ and not ‘sex’. However, we were not able to use the terminology of “cisgendered female and cisgendered male” as we did not collect the appropriate data. Therefore, in addition to the above recommended revisions, a statement was therefore also added to the Limitations section, “All studies only collected binary data on gender (i.e. male and female), thereby excluding information relating to gender non-conforming people who are disproportionately by trauma (e.g. Bauer & Scheim, 2015; Herman, Haas & Rodgers, 2014; James et al., 2016).” This statement is a small step in the journey to prevent discrimination and not to prolong the harm done through research to gender non-conforming people, by erasing them and therefore not having adequate data pertaining to them, further perpetuating health inequalities. In accordance with the gender policy of the European Association of Science Editors, Olff wrote “we need more gender- and sex-sensitive research as well as reporting” (Olff, 2017). Finally, this statement is also in line with the APA resolution on Psychology and Human Rights (APA, 2021) stating “Psychologists respect human rights and oppose the misuse of psychological science, practice, and applications and their negative impact on human rights”. Please note that I am writing these additional comments as an individual. Warmly, Dr. Shona Adams References American Psychological Association. (2015). Guidelines for psychological practice with transgender and gender nonconforming people. American Psychologist, 70(9), 832–864. https://doi.org/10.1037/a0039906 American Psychological Association (2020). Gender, Section 5.5, in Publication Manual of the American Psychological Association, Seventh Edition. https://apastyle.apa.org/style-grammar-guidelines/bias-free-language/gender American Psychological Association (2021). APA RESOLUTION on APA, Psychology, and Human Rights. https://www.apa.org/about/policy/resolution-psychology-human-rights.pdf Bauer, G. R., & Scheim, A. I. (2015). Trans people in Ontario, Canada: Statistics to inform human rights policy. http://transpulseproject.ca/wp-content/uploads/2015/06/Trans-PULSE-Statistics-Relevant-for-Human-Rights-Policy-June-2015.pdf British Psychological Society (2019). Guidelines for psychologists working with gender, sexuality and relationship diversity: For adults and young people (aged 18 and over): https://www.bps.org.uk/news-and-policy/guidelines-psychologists-working-gender-sexuality-and-relationship-diversity Docteur, E., & Berenson, R. (2010). How Will Comparative Effectiveness Research Affect the Quality of Health Care?. Urban Institute Timely Analysis of Immediate Health Policy Issues: https://www.urban.org/sites/default/files/publication/28426/412040-How-Will-Comparative-Effectiveness-Research-Affect-the-Quality-of-Health-Care-.PDF Herman, J. L., Haas, A. P., Rodgers, P. L. (2014). Suicide attempts among transgender and gender non-conforming adults. https://cloudfront.escholarship.org/dist/prd/content/qt8xg8061f/qt8xg8061f.pdf James, S., Herman, J., Rankin, S., Keisling, M., Mottet, L., Anafi, M. (2016). The report of the 2015 US transgender survey. https://transequality.org/sites/default/files/docs/usts/USTS-Full-Report-Dec17.pdf Olff M. (2017). Sex and gender differences in post-traumatic stress disorder: an update. European Journal of Psychotraumatology, 8(sup4), 1351204. https://doi.org/10.1080/20008198.2017.1351204 Steenkamp, M.M., Litz, B.T., Hoge, C.W., & Marmar, C.R. (2015). Psychotherapy for Military-Related PTSD: A Review of Randomized Clinical Trials. JAMA, 314(5), 489-500. 10.1001/jama.2015.8370 Steenkamp, M.M., Litz, B.T., & Marmar, C.R. (2020). First-line Psychotherapies for Military-Related PTSD. JAMA, 323(7), 656–657. doi: 10.1001/jama.2019.20825" } ] } ]	1	https://f1000research.com/articles/9-1252
https://f1000research.com/articles/11-153/v1	07 Feb 22	{ "type": "Case Study", "title": "Green Rating for Integrated Habitat Assessment—A green-building rating system for catalysing climate-change mitigation/adaptation in India", "authors": [ "Priyanka Kochhar", "Namrata Mahal", "Sanjay Seth", "Mandeep Singh", "Namrata Mahal", "Sanjay Seth", "Mandeep Singh" ], "abstract": "Green-building rating systems (GBRSs) are critical for implementing climate change (CC) mitigation strategies because they can help reduce greenhouse gas (GHG) emissions from the building sector. From the Indian policy perspective, the ClimateSMART Cities Assessment Framework (CSCAF) provides cities a roadmap toward mitigating CC while planning/implementing their actions and facilitates realising energy efficiency and green buildings through GBRS adoption and incentivisation. Green Rating for Integrated Habitat Assessment (GRIHA) is a comprehensive GBRS aligned with CSCAF and India’s climate goals, facilitating the implementation of Government of India’s relevant policies and climate-adaptation measures within a building project’s different phases. This paper examines existing institutional mechanisms for incentivising GRIHA-rated projects and provides recommendations for municipal bodies, regional developmental authorities, and state governments for strengthening resource efficiency in the built environment through GRIHA. Residential buildings are considered because their contribution to GHG emissions is the greatest among buildings. Data were collected through literature review, reviewing smart-city proposals and latest state annual action plans, Right to Information queries, and structured interviews of stakeholders. Feedback from green-building certification agencies, project proponents, and government officials revealed a need for local-level information dissemination and guidance on institutional mechanisms for incentivising green-rated projects. Further, to understand the implementation mechanisms for GRIHA-linked incentives, residential projects under some local-government agencies were documented as case studies, providing useful insights into prevalent mechanisms for availing incentives while facilitating GRIHA compliance. The information provided herein can be useful for local governments in other developing countries for guiding the building sector toward mitigating climate change.", "keywords": [ "Green-building rating systems", "green-building policies", "green-building incentives", "climate change mitigation/adaptation", "Green Rating for Integrated Habitat Assessment (GRIHA)", "SDG11 Sustainable Cities and Communities", "SDG6 Clean Water and Sanitation", "UN Sustainable Development Goals", "SDG13 Climate Action", "SDG17 Partnerships to achieve the Goal" ], "content": "1. Introduction\n\nBuildings contribute to 39% of all energy-related CO2 emissions (UN Environment and the International Energy Agency, 2017), of which, operational emissions (emissions from energy used for heating, cooling, and lighting buildings) account for 28% and the remaining 11% is from embodied carbon emissions associated with materials and construction processes throughout the entire building lifecycle (World Green Building Council, 2019). With the world’s population projected to be mostly urban by 2050 (United Nations, 2018), this contribution will rise with rapid economic growth, particularly in developing countries. Therefore, for increasing stakeholders’ awareness, publishing information regarding institutional mechanisms for implementing green-building rating system (GBRS)-linked incentives in the built environment is critical for expediting informed decision making.\n\nAccording to the World Resources Institute (WRI) (WRI, 2020), the top 10 emitting countries (Figure 1) account for more than two-thirds of annual global GHG emissions: China leads with 26.1%, followed by the United States (12.67%), the European Union (7.52%), and India (7.08%). Moreover, residential buildings are the top contributors among buildings (Figure 2) (WRI, 2020).\n\nGBRSs are the ideal tool for validating buildings’ sustainability claims by setting performance benchmarks to promote improved construction and operational resource efficiency (Ramkumar, 2020). Although various GBRSs exist globally, tailoring them to suit local ecosystems, regulations, and requirements in developing countries (e.g., India) is important. Green Rating for Integrated Habitat Assessment (GRIHA) was India’s first indigenous GBRS endorsed by the Government of India (GOI) and showcased at the United Nations Framework Convention on Climate Change (UNFCCC) as part of various initiatives for meeting India’s climate goals (UNFCC, 2015; Ministry of New and Renewable Energy (MNRE), GOI, 2019). Although GRIHA is over a decade old, further dissemination of information regarding its ground implementation in different regions of India is required. In particular, guidelines are required for states, municipalities, and development authorities for formulating, endorsing, and enforcing green initiatives and policies to support India’s CC commitments.\n\nThe ClimateSMART Cities Assessment Framework (CSCAF) is aimed to guide cities toward climate actions and help make them more responsive and less vulnerable to CC. CSCAF’s “Energy and Green Buildings” category includes six important indicators, including “Promotion of green buildings” and “Green Building Adoption” (National Institute of Urban Affairs, 2019), which play an important role in CC mitigation and adaptation. Because domestic and commercial sectors consume approximately 33% of electricity (Ministry of Statistics and Programme Implementation, GOI, 2019; Bureau of Energy Efficiency (BEE), GOI, 2020) in India, significant untapped potential exists for realising highly energy-efficient and resilient buildings through the implementation of green-building policies and programs. Multiple documents (Climate Centre for Cities, 2020) provide information and examples from cities, international cases, and other organisations; however, only one relevant report—prepared by WRI India to address the requirements of the above-mentioned indicators (WRI India, 2020)—serves as a guidebook for local governments for green-building policy design and implementation. The report provides guidance on developing and implementing two specific types of building efficiency actions: green-building codes and certification/rating systems. Along with documenting the implementation of Energy Conservation Building Code (ECBC) in Hyderabad, the report provides information on implementing Pune Municipal Corporation’s Eco-Housing Program, which is neither active nor a part of CSCAF. However, it lacks suggestions on implementing GBRSs suggested in CSCAF (namely, Leadership in Energy and Environmental Design (LEED), Excellence in Design for Greater Efficiencies (EDGE), Green Rating for Integrated Habitat Assessment (GRIHA), Indian Green Building Council (IGBC) GBRSs, and Green and Eco-friendly Movement (GEM)) or provide information on current institutional mechanisms that may be replicated/adopted or adapted by other cities. To address the mentioned gap, this paper examines existing institutional mechanisms for incentivising GRIHA-rated projects and provides recommendations for municipal bodies, regional developmental authorities, and state governments to improve CSCAF rankings by strengthening resource efficiency in the built environment though GRIHA to facilitate the implementation of various policies and schemes of GOI according to its international green commitments.\n\nFurther, case studies were conducted at the municipal and development-authority levels to study GRIHA’s implementation according to the institutional mechanisms in place for availing incentives to provide improvement recommendations. This study aims to provide a comprehensive knowledge base, data, and guidelines for formulating and implementing policies, recommendations, and initiatives for boosting India’s green-building efforts in the built environment, which constitute a vital part of the global efforts toward CC mitigation and adaptation. Moreover, this paper can provide a reference for local governments in other developing countries and help them in creating frameworks for CC mitigation and adaptation by guiding the building sector toward green-building practices.\n\n\n2. Methods\n\nThe methodology adopted to accomplish the objectives of this study included literature review, exhaustive official-documentation review, stakeholder identification, designing of research tools such as structured interviews of the stakeholders identified, data collection and analysis, and formulating guidelines for effectively implementing green-building schemes.\n\nLiterature and official documentation reviews for an in-depth understanding of linkages among CC, India’s building sector, and India’s Intended National Determined Contributions (INDCs) were conducted. To understand the city-level GBRS penetration level in the policy framework, smart-city proposals of all 100 cities under the Smart Cities Mission (SCM) (described in Section 4) and state annual action plans (SAAPs) prepared under the Atal Mission for Rejuvenation and Urban Transformation (AMRUT) for all 36 Indian states and union territories (UTs) were reviewed (see Extended data) (Kochhar, 2022). Furthermore, GRIHA and IGBC websites were studied to map information on GBRS-linked initiatives (including incentives, disincentives, and mandates) offered by various Indian states and UTs. Information on government-linked incentives for LEED, EDGE, and GEM is not publicly available on their websites and hence could not be included herein. The 100 Smart City proposals were reviewed to evaluate whether cities included GBRSs as a tool to achieve essential features of SCM guidelines (i.e., at least 80% buildings under redevelopment and green field should be energy efficient and green buildings) in area development plans (Smart Cities Mission Statement and Guidelines, May 2015). The latest SAAPs (see Extended data) (Kochhar, 2022) were reviewed to evaluate whether the proposed/completed reforms for conducting energy and water audits included green building incentives (e.g., rebate in property tax or charges connected to building permission/development charges) (Ministry of Housing and Urban Affairs (MoHUA), GOI, 2015). Information on green-building incentives, which is available online (GRIHA, 2020; Indian Green Building Council, 2015; Administrative staff College of India and Natural Resources Defense Council, 2014) and through notifications, was analysed in detail. A Right-to-Information (RTI) query was filed with the Town and Country Planning Department, GOI, to collect information on states that have integrated GBRSs in their building byelaws (RTI query, 2020). Structured interviews were conducted to identify challenges being faced by stakeholders that are awarding and availing incentives in states and municipal corporations with the highest number of policies and registered/green-rated projects. Several stakeholders, including municipal officials, private developers, project consultants, and residents of GRIHA-rated projects in New Okhla Industrial Development Authority (Noida) and Pimpri Chinchwad, were interviewed for case-study documentation.\n\nBecause GRIHA has established itself as the national rating for green buildings and drives policy compliance in India, states with a prominent GRIHA footprint, including Uttar Pradesh (UP) (7% of GRIHA project) and Maharashtra (40% of GRIHA projects), were shortlisted to identify case studies on the institutional mechanisms in place.\n\n\n3. Role of various rating systems in mitigating climate change in India and abroad\n\nGBRSs provide a framework for designing, implementing, and monitoring energy efficiency and CC mitigation and adaptation measures. Some green-design benefits to a building owner, user, and the society overall are as follows:\n\n• Reduced energy consumption without sacrificing comfort levels\n\n• Reduced destruction of natural areas, habitats, and biodiversity, and reduced soil loss from erosion\n\n• Reduced air and water pollution (with direct health benefits)\n\n• Reduced water consumption\n\n• Limited waste generation due to recycling and reuse\n\n• Reduced pollution loads\n\n• Increased user productivity\n\n• Enhanced image and marketability\n\nRobust implementation of existing green-building policies and formulation of new interventions in the building sector at the national, state, and municipal levels (Colenbrander et al., 2016), including by providing incentives for GBRSs, continue to be crucial in achieving India’s INDCs. INDCs represent the principal mode for governments to internationally communicate their intended steps to deal with CC in their countries (WRI, 2021). In anticipation of the historic Paris Agreement in 2015 (UNFCC, 2021), countries publicly outlined their intended post-2020 climate actions under the new international agreement, which were called their INDCs (WRI, 2021). GRIHA has been acknowledged in India’s INDCs (UNFCC, 2015).\n\nIn the Indian and global contexts, several GBRSs exist (e.g., IGBC GBRSs, GEM, CASBEE, BCA Green Mark, BREEAM, LEED, and EDGE). Globally, voluntary building-rating systems have been vital in increasing awareness and promoting green design. However, most of them were tailored according to the building industry of their origin country.\n\nIndia’s dynamic population and economic growth have resulted in a tremendous demand and construction of buildings, thereby exerting enormous pressure on resource availability. To achieve sustainability, policy makers are addressing the environmental pressures of increased resource demand coupled with a rapidly changing climate at different levels. Various policies and regulatory mechanisms have been devised and implemented through national plans and programs to address urban challenges. GOI ministries and agencies have designed frameworks, codes, and standards such as National Building Code (NBC) (SP 7: 2016 National Building Code of India 2016 (NBC 2016), December 14, 2018) for regulating building construction activities across India; Environmental Clearance (Ministry of Environment, Forests, and Climate Change (MoEFCC), 2014) to ensure resource-use efficiency for large projects (>20,000-m2 built-up area); ECBC (A Report on Impact of Energy Efficiency Measures For the Year 2018–19, March 2020), which is applicable to air-conditioned commercial buildings with a connected load of >100 kW; the Solar Buildings Programme for Energy-Efficient Buildings (MNRE, GOI, 2019) for implementing renewable energy in buildings; AMRUT (Thrust Areas, October 25, 2021) for ensuring sufficient robust sewage networks and water supply; and SCM (About Smart Cities Mission, 2021) for promoting sustainable and inclusive cities. However, the greatest challenge arguably is building capacity and skills among stakeholders to devise solutions, effective ground implementation of such initiatives at the local level, and optimising their effectiveness. This could be achieved by encouraging a more holistic approach to building. Examples of some difficulties faced during the implementation of sustainable-habitat policies are the lack of disincentives for noncompliance, agencies and systems working in factions (i.e., different departments at the central and state governments independently examining areas such as energy efficiency, renewable energy, water resources, and waste management, contrary to a holistic approach that would address the building sector encompassing water, energy, etc. all together), and executing policies based on codes and standards before on-site verification, leading to on-site implementation challenges. Considering such challenges, and with the overall objective of reducing resource consumption and GHG emissions as well as enhancing renewable and recycled resource use by the building sector, The Energy and Resources Institute (TERI) developed GRIHA in 2005, which was adopted by the Ministry of New and Renewable Energy (MNRE), GOI, in 2007 (MNRE, GOI, 2019; MNRE, GOI, 2017; MNRE, GOI, 2020). GRIHA was also developed to indigenise design and implementation of resource efficiency for buildings in India because most international GBRSs did not cater to the Indian real-estate sector’s requirements in the 2000s. The GRIHA rating framework is an evaluation tool for measuring and rating a building’s environmental performance, facilitating design, and evaluating a project throughout its lifecycle, including pre-construction, building planning and construction, and operation and maintenance stages. In addition to reducing the GHG emission from buildings, GRIHA optimises electricity consumption while meeting comfort requirements and reduces dependence on fossil fuel-based electricity and stress on natural resources. Other benefits of GRIHA-rated buildings include reduced air and water pollution, optimised water consumption, and waste management. GRIHA is based on a star rating system with a certain number of points required to achieve a particular star rating. A project must achieve minimum 50 points to qualify for GRIHA rating. A project’s scoring to achieve GRIHA rating is as follows (Kochhar and Singh, 2021):\n\n• 50–60 points: GRIHA 1-Star rated\n\n• 61–70 points: GRIHA 2-Star rated\n\n• 71–80 points: GRIHA 3-Star rated\n\n• 81–90 points: GRIHA 4-Star rated\n\n• 91–100 points: GRIHA 5-Star rated\n\nAs a tool, GRIHA includes qualitative and quantitative assessment criteria, which facilitate rating a building based on its level of “greenness”. GRIHA endeavours to minimise a building’s resource consumption, waste generation, and overall ecological impact to within specific nationally acceptable limits/benchmarks. Figure 3 shows GRIHA’s evolution.\n\n3.1.1 Why GRIHA?\n\nGRIHA was the first GBRS endorsed by GOI. At the national level, MNRE incentivised GRIHA in 2007 and provided subsidies to projects by covering registration and certification fees (Ministry of Non-conventional Energy Sources (Urban, Industrial, and Commercial Group), GOI Notification, 2009), while internationally, MoEFCC presented GRIHA as the national rating system as part of India’s INDCs to UNFCCC for the Paris Convention. GRIHA was the India’s first GBRS to be adopted and adapted by the Central Public Works Department (CPWD) (CPWD Office Memorandum, 2009), an agency under MoHUA; CPWD’s green-building schedules and specifications were also formulated according to GRIHA (TERI, 2012). Further, GRIHA aligns itself with Indian codes and standards and, in 2006–07, was uniquely equipped to share feedback with BEE for ECBC compliance and with MNRE for renewable-energy installation. Over time, other rating systems have also emphasised on local-code compliance and been endorsed by various government agencies.\n\nOn a broader scale, GRIHA, in conjunction with associated activities and processes, will benefit the community overall with an improvement in the environment by reducing GHG emissions, energy consumption, and the stress on natural resources.\n\n3.1.2 GRIHA as an implementation tool for climate-change mitigation policies\n\nGRIHA attempts to quantify aspects such as energy consumption, waste generation, and renewable energy adoption to manage, control, and optimise them.\n\nAs an example (Construction World Staff, March 1, 2014), 10 million m2 of a GRIHA 5-star-certified project can save sufficient electricity to power around 100,000 urban homes, save enough water to meet the needs of 22,000 urban homes, afford 6-MW photovoltaic (PV) installation to enhance electricity supply, and provide monitored data to ensure and strengthen compliance (Construction World Staff website, March 1, 2014).\n\nAs per the Efficient (EFF) scenario modelled by TERI (TERI, 2018), efficient space conditioning, urban and rural lighting, and refrigeration in the residential sector can potentially reduce energy intensity by up to 10% by 2031 (Figure 4a). By using the same EFF scenario, higher penetration of GRIHA-rated buildings in the commercial sector can potentially achieve up to 15% energy-intensity reduction by 2031 (Figure 4b).\n\nBesides being a tool that addresses CC mitigation through the evaluation of environmental performance of a building throughout its lifecycle, GRIHA addresses and alleviates risks caused by extreme heat, water scarcity, and flooding. GRIHA criteria embed CC-adaptation measures through the design, construction, and operational phases of building projects (GRIHA Council, 2020). Almost 50% of the points awarded in the GRIHA system have the potential to contribute toward CC adaptation and resilience as described below:\n\nMeasures for adaptation to extreme heat\n\n• Low-impact design: Natural ventilation/low-energy cooling systems.\n\n• Design to mitigate urban heat island intensity: Surfaces are soft paved/covered with high-solar-reflective coating, shaded by trees, pergolas, and/or solar panels.\n\n• Preservation and protection of landscape (native/naturalised tree planting) during construction.\n\n• Energy efficiency: Minimise overall heat gain and reduce energy demand for cooling.\n\nMeasures for adaptation to flooding\n\n• Storm water management: 100% post-construction stormwater runoff management on site and scheme to deliver harvested rainwater to users.\n\nMeasures for adaptation to water scarcity\n\n• Efficient water use during construction: Use of treated wastewater/captured rainwater in construction activities; strategies to reduce water use.\n\n• Optimisation of building and landscape water demand: Installation of water-efficient systems such as low-flush toilets, use of regionally appropriate xerophyte plant species, and efficient landscape irrigation systems.\n\n• Water reuse: Tertiary-level wastewater treatment for 100% of sewage generated on site, artificial groundwater recharge and rainwater storage, and maximum utilisation of treated and harvested water within the site to reduce the complete dependence on fresh water supply.\n\nMeasures to enable behaviour change by increasing awareness levels\n\n• Create environmental awareness among building occupants and invitees.\n\nOther measures\n\n• Site plan must conform to the development master plan; provisions of ecosensitive, coastal, and heritage zones away from water bodies; and meeting “various hazard-prone area regulations”\n\n• Strategies independent of other criteria that make the project more sustainable\n\n• Quality of water available for use during the operational phase of the building meets national standards.\n\nGraham and Rawal (2019) stated that GBRSs in India are not yet key to achieving the emission reduction potential in India; however, they recommended that achieving the potential through effective implementation would be important toward the national effort.\n\n3.1.3 ClimateSMART Cities Assessment Framework\n\nTo mitigate CC impacts, MoHUA launched the Climate Smart Cities Assessment Framework (CSCAF) in 2019 for 100 Smart Cities (MoHUA Press Release, 2020). CSCAF, which is a culmination of the major CC mitigation and adaptation schemes of GOI, facilitates the realisation of energy efficiency and green buildings through GBRS adoption and incentivisation. CSCAF’s objective is to provide cities a clear roadmap toward mitigating CC while planning and implementing their actions, including investments (MoHUA Press Release, 2020). CSCAF has 28 indicators across five categories (Figure 5) to assist cities in understanding CC-linked challenges and improvement areas. Because GBRSs facilitate energy efficiency implementation (among other benefits), the CSCAF for 100 smart cities (MoHUA, GOI, 2019) facilitates the inclusion of GBRSs, including star labelling for buildings by GRIHA, EDGE, LEED, IGBC, and GEM. CSCAF also serves as a culmination of other schemes such as Green India Mission, National Clean Air Programme, AMRUT, and Swachh Bharat Mission in achieving the objective of being “Climate Smart.”\n\nIn the first phase of city assessment according to CSCAF, 96 cities including more than 27 government departments/organisations from a three-tier governance structure—national, state, and city—along with other stakeholders participated and provided inputs for >120 datasets. Cities submitted data on the official portal; an Expert Committee evaluated these submissions. With an intent to inform cities on their climate readiness, the first baseline assessment for each city was announced.\n\nInformation from knowledge-sharing platforms revealed that cities were learning from each other’s experiences and were motivated to work toward tackling CC impacts collectively. The success stories, best practices, advisories, and other reference material from the first assessment are available on SmartNet (ClimateSmart Cities, 2019) to help other cities in their endeavour.\n\n\n4. Key climate-change initiatives by Indian government agencies for climate-change mitigation and adaptation through the built environment\n\nDifferent GOI ministries, such as the Ministry of Power (including Energy Efficiency Services Limited and BEE); MNRE; Ministry of Housing and Urban Poverty Alleviation (including CPWD; Bureau of Indian Standards); and MoEFCC, have played a key role in designing policies and incentives for projects to incorporate resource efficiency through GBRSs, in turn meeting India’s INDCs (Figure 6).\n\nWith an objective to drive economic growth and improve people’s quality of life by enabling local-area development and harnessing technology, especially technology that leads to “smart” outcomes, GOI launched SCM in 2015 (About Smart Cities Mission, 2021). In the Indian context, the smart-cities concept is based on six fundamental principles: (i) communities at the core of planning and implementation; (ii) ability to generate greater outcomes using lesser resources; (iii) cities selected through competitions with flexibility to implement projects; (iv) innovating methods for integrated and sustainable solutions; (v) careful selection of technology, relevant to the cities’ context; and (vi) sectoral and financial convergence (About Smart Cities Mission, 2021). SCM envisions developing areas within 100 cities in India as model areas based on an area-development plan. Each city has a mandate to create a special-purpose vehicle, presided by a full-time chief executive officer, to implement SCM. Cities are selected in two stages: (1) 100 smart cities are distributed among the states and UTs based on unbiased criteria and (2) each potential city prepares its Smart City Proposals, which contain the model chosen (retrofitting or redevelopment or greenfield development or a mix thereof) in addition to a pan-city dimension (100 Smart Cities Mission, 2021). According to information available on the MoHUA website (About Smart Cities Mission, 2021) Rs. 2,005,018-million investment is proposed across 100 cities and projects worth Rs. 531,760 million have been completed (About Smart Cities Mission, 2021). Along with SCM, AMRUT (The Mission, 2015) was launched in 2015 focusing on establishing infrastructure for ensuring sufficient robust sewage networks and water supply for urban transformation by implementing urban revival projects. In 2015, the Union Ministry of Urban Development announced the Smart Cities Challenge (SCC) (100 Smart Cities Mission, 2021), which aims to create exemplar areas in cities by merging innovative plans with latest technologies. By creating these replicable exemplar areas, SCC aims to redefine the way a city is imagined in India. SCC intends to find solutions to major problems in cities by merging innovative plans and latest information technologies, improve economic opportunity and quality of life, and ensure public accountability. As per the city challenge reports submitted (City Challenge, 2021), only 25 out of 100 cities have included green-building certification as a means to achieve select essential features, requiring “At least 80% buildings (in redevelopment and green field) ought to be energy efficient and green buildings” (Smart Cities Mission Statement and Guidelines, 2015). Twenty-two SAAPs submitted as a part of AMRUT (SAAP 2017–2020, 2020) attempt to “award incentives for green buildings (e.g., rebate in property tax or charges connected to building permission/development charges)” (SAAP - Atal Mission for Rejuvenation and Urban Transformation, 2015). Furthermore, Andaman and Nicobar Islands, Andhra Pradesh, Chhattisgarh, Himachal Pradesh, Jharkhand, and UP have issued directions to incentivise green-rated buildings, as reported in their respective SAAPs (SAAP 2017–2020, 2020).\n\nThe use of GBRSs as implementation tools for AMRUT and SCM facilitates a bottom-up approach, thereby enabling improved transparency and efficiency between state governments and municipal bodies. Fifteen states and UTs—Andhra Pradesh, Chandigarh, Chhattisgarh, Gujarat, Haryana, Karnataka, Kerala, Maharashtra, Manipur, Delhi, Punjab, Telangana, UP, Uttaranchal, and West Bengal—have endorsed GBRSs as part of their revised or updated building byelaws (RTI query, 2020). Smith and Pathak (2018) have identified GBRSs as effective tools to achieve urban transformation at the level of urban local bodies by implementing relevant components of SCM (Smart Cities Mission Statement and Guidelines, 2015) and AMRUT.\n\nFeedback from certification agencies, project proponents, and government officials has indicated that comprehensive information dissemination on institutional mechanisms is needed for municipal bodies to integrate GBRSs within their existing framework.\n\nIn India, green buildings are generally perceived as more cost intensive than regular buildings, which is a major barrier in their extensive adoption. This challenge is particularly pronounced in residential buildings, where private developers bear the incremental costs and occupants reap the recurring benefits of low operating costs. Table 1 (MoHUA, GOI, 2019) presents percentage savings in resource consumption achieved by green-rated buildings versus conventional buildings and indicative increase in cost of green-rated buildings, which may (in part) be set off by incentives.\n\nAt the state and municipal levels (Table 2), 17 States and UTs (out of 36 States and UTs (States and Union Territories, 2021) have incentivised (by providing financial incentives, additional ground coverage, additional floor area ratio (FAR)/floor space index (FSI)/building area ratio (BAR)) or issued direction for mandatory compliance with various GBRSs to achieve goals set at the national level, which facilitate and contribute to achieving INDCs.\n\nGBRSs in India provide implementation mechanisms for national- (Figure 4), state-, and local-level policies (Table 2) to achieve resource efficiency in the built environment (Knowledge Repository, 2021; Lok Sabha Questions, December 13, 2018; Kochhar, 2010). They also facilitate building performance monitoring and quantification (Graham and Rawal, 2019), which supports capture, management, and public availability of data toward reporting of India’s INDCs. GBRSs are embedded in local-level policies, while agencies mandate GBRSs through integration in building byelaws and provide GC and FAR incentives. GBRSs (in general) and GRIHA for new and existing buildings (in particular) incorporate ECBC and the Standards and Labelling program for appliances (issued by BEE, Ministry of Power), which are closely aligned with the environmental-clearance norms for buildings issued by MoEFCC and mandate renewable-energy integration (aligned with MNRE objectives) (Ministry of Environment and Forests (MoEF), GOI Office Memorandum, 2011). Furthermore, GBRSs are also aligned with the National Action Plan on Climate Change, incorporate relevant sections of NBC, and facilitate partial implementation of SCM and AMRUT, which are important initiatives of MoHUA. Considering the above-mentioned alignment with the objectives of GOI ministries, GBRSs have been embedded in local-level policies to ascertain action toward CC mitigation and adaptation. Nine local bodies mandate GBRSs through their integration with building byelaws, 21 provide ground coverage and/or FAR incentives, and six provide financial incentives. Table 2 provides information on the local bodies that have employed GBRSs to achieve CC mitigation and adaptation goals.\n\nTable 2 provides cities and municipalities a roadmap that can help to enhance their scores based on the CSCAF framework through implementation of green-building policies and incentives.\n\n\n5. Methods of meeting GRIHA requirements according to CSCAF\n\nGreen-rated project incentives provide an institutional framework at the state and municipal levels to implement policies, including the CSCAF developed for 100 Smart Cities, and enable cities to achieve the objective of being “Climate Smart.”\n\nUP (nine policies), Gujarat (seven policies), Maharashtra and Punjab (six policies each), and Andhra Pradesh and West Bengal (five policies each) are states (Table 3) that utilise GBRSs, particularly GRIHA, to implement CC mitigation, adaptation, and resilience policies (GRIHA Council, 2020).\n\nNoida in UP with more than 100 green buildings (M. Goyal, personal communication, September 1, 2020), Pimpri Chinchwad Municipal Corporation (PCMC) with over 60 GRIHA projects (K. Karmarkar, personal communication, September 1, 2020), and the state of Haryana with over 30 green buildings (H. Singh, personal communication, September 1, 2020) are eligible to receive the green rating-linked additional FAR incentive.\n\nSelect GRIHA projects in the above-mentioned regions were identified for supporting information on implementation mechanisms. Developers, namely Goel Ganga Developers (A. Goel, personal communication, August 28, 2020) and Sanjeevani Developers in Pimpri Chinchwad (S. Deshpande, personal communication, August 29, 2020) and the DAH group in Noida, were interviewed (S. Sadarangani, personal communication, August 30, 2020) to gain information on the role of incentives in offsetting any additional costs incurred by the developers in the design and construction of green buildings. Occupants of the GRIHA-rated Ganga Skies project were interviewed (Mahal, 2016) to understand the impact of property tax rebates during residence in a green building.\n\n5.2.1 GRIHA-linked incentives by PCMC\n\nIn 2011, PCMC launched GRIHA-linked incentives under a scheme called “energy-efficient solar/green buildings” by MNRE (MNRE, GOI report, 2018). According to the incentive scheme (Table 4), and depending on the level of GRIHA rating, the project developer is eligible to avail 10%–50% discount in Premium1 and occupants are eligible to avail property tax benefit (between 5% and 10%) based on the final rating (Green Building Initiative, 2021). Furthermore, according to the office memorandum released by the MoEF, GOI (MoEF, GOI Office Memorandum, 2011), a GRIHA pre-certified project would be eligible for “out-of-turn priority attention by the Expert Appraisal Committee (EAC)/State-Level Expert Appraisal Committee (SLEAC).”\n\n5.2.2 Operationalisation of GRIHA-linked priority consideration by EAC/SLEAC\n\nThe following steps need to be followed to operationalise GRIHA-linked priority consideration for a project by EAC/SLEAC:\n\n• Register the project at www.grihaindia.org\n\n• Submit a proposed development plan for the registered project with recent date-stamped site photographs\n\n• GRIHA Council to review feasibility\n\n• Successful payment of registration fee to access the GRIHA Online Panel\n\n• Project team to upload compliance documents through the online panel\n\n• Submitted documents evaluated by GRIHA Council\n\n• GRIHA Council to revert with comments on the submitted documents within two weeks\n\n• Revised documents to be submitted by the clients within two weeks of receiving comments\n\n• Documents evaluated by a pre-certification committee\n\n• In case of compliance, pre-certification is awarded along with a detailed compliance report\n\n• In case of non-compliance, a report with necessary corrective actions is shared\n\n• GRIHA registration of project is mandatory after pre-certification\n\n• GRIHA rating to be pursued as per GRIHA Council procedure\n\n5.2.3 Operationalisation of GRIHA-linked incentives at PCMC for project developers\n\nThe process to avail GRIHA incentives under PCMC (Green Building Initiative, 2021) is appended below:\n\n• Register project at www.grihaindia.org\n\n• Payment of registration fee and access to GRIHA Online Panel\n\n• One-day GRIHA workshop for project team by GRIHA Council\n\n• First due-diligence site visit conducted by GRIHA Council (when project construction is at plinth level) and report (with feedback) uploaded online for project team\n\n• Compliance report submitted by project team within 15 days of the first site visit\n\n• Second due-diligence site visit conducted by GRIHA Council upon completion of superstructure and second report uploaded online for the project team\n\n• Compliance report submitted by project team within 15 days of the second site visit\n\n• Project team to upload GRIHA documentation for all criteria\n\n• Primary evaluation by GRIHA Council on completeness of the documentation\n\n• Third and final due-diligence site visit conducted by GRIHA Council\n\n• GRIHA evaluation committee reviews GRIHA documentation for award of provisional points\n\n• Project evaluation report uploaded online. Project team may submit additional documents within 30 days of uploading evaluation report.\n\n• GRIHA provisional rating and certificate issued by GRIHA Council\n\n• Post-occupancy audit conducted by BEE-certified auditor\n\n• Award of final GRIHA rating after building is fully commissioned and is operational for at least 12 months.\n\n• Developer to submit GRIHA rating certificate to PCMC for release of rebate on Premium. Environment Cell of PCMC notifies tax department to issue applicable rebate in Premium.\n\n• Tax department to release rebate on property tax for occupants of the property for three consecutive years, after which the building to be re-audited for energy, water, and waste (report to be prepared by BEE-certified energy auditor) every three years to avail the property tax benefit.\n\n5.2.4 Impact of incentives on PCMC\n\nBased on information from PCMC (K. Karmarkar, personal communication, September 1, 2020), around 60 projects registered till 2019 are eligible to avail incentives. It had been inferred (Green Building Initiative, 2021) that on an annual basis, if the PCMC revenue from Premium is Rs. 1 billion2, then the revenue invested for 3-Star GRIHA projects through rebates would be approximately Rupees (Rs). 40 million. In turn, a 35% reduction in the quantity of potable water required, 35% reduction in the amount of wastewater generated, and 15% of treated wastewater to be used for various applications in new buildings are expected, thereby reducing the pressure of providing services and maintaining infrastructure.\n\nIn the case of property tax rebate for a typical case, where the end user pays an annual base tax of Rs. 6000/- for a 92.9-m2 flat, a rebate of Rs. 600/- per annum would be available for a 3-Star GRIHA project (Green Building Initiative, 2021). Similarly, for a 4645.2-m2 property (around 50 flats) and a 37,161.2-m2 property (around 400 flats), a tax rebate of Rs.30,000/year and Rs. 240,000/year would be applicable, respectively. In turn, the project would benefit through rainwater harvesting, solid waste management, and availability of solar thermal systems on site.\n\n5.3.1 Green-building rating-linked incentives by Noida\n\nIn 2010, as part of the General Provisions for building projects, Noida included additional free FAR for LEED-certified projects constructed on plot sizes of >5000 m2 (NOIDA and Greater NOIDA embrace GRIHA. GRIHA incentives, October 20, 2012; M. Goyal, personal communication, September 1, 2020). Subsequent amendments in 2012 and 2019 included incentives for GRIHA- and IGBC-certified projects as well.\n\n5.3.2 Operationalisation of green building rating-linked incentives at Noida for project developers\n\nThe process to avail green building-linked incentives in Noida (NOIDA and Greater NOIDA embrace GRIHA. GRIHA incentives, October 20, 2012; M. Goyal, personal communication, September 1, 2020) is appended below:\n\n• Applicant submits LEED/GRIHA/IGBC pre-certification to the Town Planning Department of Noida to seek 5% additional free-of-cost FAR for green building.\n\n• At the time of seeking a completion certificate, the applicant to provide final LEED/GRIHA/IGBC rating certificate to Noida.\n\n• The applicant is required to submit a rating certificate and a certificate of compliance every five years. In case the client fails to do so, the authority—after one month’s notice—may charge the compounding fees of the FAR given free of cost at a rate of 200% of the cost of purchasable FAR.\n\n5.4.1 Green-building rating-linked incentives by Haryana\n\nWith an objective to synchronise the provisions of building rules across the state, the Government of Haryana issued the Haryana Building Code in 2017 (The Haryana Building Code 2017, 2021), which incorporates the requirements of the Model Building Byelaws (Model Building Bye Laws, 2016).\n\nThe Haryana Building Code provides green-building measures and incentives (for new buildings) where projects certified as green buildings are eligible for additional FAR incentives (Table 5). Furthermore, “in case the building is certified from GRIHA, there is no requirement for issuing environmental clearance” (MoEF, GOI Office Memorandum, 2011). The applicant only has to pay the Infrastructure Development Charges on additional FAR granted as incentive under Code 6.5, i.e., green-building measures and incentives, and not the fee for availing extra FAR (H. Singh, personal communication, September 1, 2020).\n\n5.4.2 Operationalisation of green building rating-linked incentives in Haryana\n\nThe process to avail green building rating incentives in Haryana (The Haryana Building Code 2017, 2021; H. Singh, personal communication, September 1, 2020) is appended below:\n\n1. Applicant is required to submit provisional certificate from GRIHA or precertification from IGBC/LEED at the time of seeking approval of building plan, to the Competent Authority.\n\n2. Applicant is required to submit final rating certificate from GRIHA/IGBC/LEED at the time of applying for the Occupation Certificate of building.\n\n3. Competent Authority verifies final level of rating achieved with the provisional/precertification rating and issues Occupation Certificate, and approves claim of additional FAR if final rating is same/higher than the provisional rating.\n\n4. In case the final rating achieved is lesser than the provisional rating, occupation certificate is issued after compounding the additional FAR (i.e., difference of additional FAR from provisional rating and final rating) acquired by the applicant, at 10 times the rates of External Development Charges (EDC) (The Haryana Building Code 2017, 2021) applicable at the time of submission of occupation certificate application.\n\n5. The applicant to submit a rating certificate for the building from GRIHA/IGBC/LEED every 5 years. In case of non-compliance, the authority, after giving one month's notice may charge compounding fee or may take appropriate action on a case-by-case basis.\n\n\n6. Case studies\n\nTo evaluate GBRS implementation and its outcome in residential projects, case studies were conducted for some projects under two municipal corporations and one development authority. The following subsections are dedicated to these case studies.\n\nThe 3-Star GRIHA-rated residential project (Figure 7) (56,000-m2 built-up area) availed 30% rebate in Premium. The occupants were entitled to a 5% property tax rebate. In addition, the project could achieve 8% reduction in capital cost by integrating green-building principles at appropriate design, planning, and implementation stages (Mahal, 2016).\n\nThe environmental benefits accrued by the project as part of GRIHA (Construction World Staff, March 1, 2014) are listed below:\n\n• 30% reduction in energy consumption compared with the GRIHA benchmark.\n\n• 25% reduction in building water consumption.\n\n• 40% reduction in landscape water consumption.\n\n• More than 50% of the living areas are day-lit.\n\n• More than 40% fly-ash used in the block work.\n\n• 10-kW renewable energy generated on site.\n\nThe 3-Star GRIHA-rated residential project (Figure 8) of 3100-m2 built-up area (Complete, GRIHA, Projects, Pune, Residential. Devrai, Phase II, September 2019) availed 30% rebate in Premium. The occupants were entitled to a 5% property tax rebate, resulting in annual savings (Incentive release approval for GRIHA rated project by PCMC, September 2019; GRIHA Council organised “Paryavaran Rakshak” programme for occupants of Devrai Phase-II, Pune, September 21, 2019).\n\nThe environmental benefits accrued by the project as part of GRIHA are listed below (Complete, GRIHA, Projects, Pune, Residential. Devrai, Phase II, September 2019):\n\n• 85% estimated reduction in energy consumption compared with the GRIHA benchmark.\n\n• 76% water savings in landscape usage due to drip irrigation installation.\n\n• More than 90% of interior day-lit spaces.\n\n• Solar hot-water system to meet 96% of hot water requirement, thus reducing the consumption of energy generated from non-renewable sources.\n\n• 3-kWp solar photovoltaics installed for common-area lighting.\n\n• 29% reduction in embodied energy in structural application.\n\nThe GRIHA pre-certified mixed-use project of approximately 338,402.6-m2 built-up area (Figure 9) (S. Sadarangani, personal communication, September 12, 2020) and cost of Rs. 8000 million (View Projects, September 14, 2020) availed 5% additional free-of-cost FAR of 11,745 m2. The cost of additional free-of-cost FAR provided to the project (calculated according to the compounding fee letter of Noida (M. Goyal, personal communication, September 1, 2020)3, using rate of purchasing commercial/residential FAR) is approximately Rs. 768 million to the developer. The projected incremental cost (as the project is still under construction) incurred to meet the requirements of GRIHA 4 Star (including solar reflectance index tiles, fly-ash bricks, double glazing, roof insulation, BEE-rated ceiling fans, solar PV power plant, and HVAC automation) is approximately Rs. 694.5 million (S. Sadarangani, personal communication, September 12, 2020). Thus, the additional cost incurred by the project is absorbed by the value of the free FAR incentive for GRIHA projects provided by Greater Noida.\n\n6.3.1 Impact of incentives on NOIDA\n\nBased on information from Noida (M. Goyal, personal communication, September 1, 2020), over 100 projects (i.e., almost 80% of total eligible projects (plot size of >5000 m2) that apply for building permission) have registered between 2010 and 2020 with LEED/GRIHA/IGBC to avail the additional free-of-cost FAR incentive awarded based on byelaw provisions by the authority. This initiative has encouraged private developers to adopt green-building practices, with negligible impact on Noida’s revenues. Numerous group-housing projects have applied for the incentive, but because almost 90% of the projects are partly complete, the final sanction is awaited.\n\n\n7. Discussion\n\nStates, particularly where SCM and AMRUT schemes are being actively implemented, are able to ensure resource efficiency through GBRSs (Table 2), e.g., UP, Haryana, and Maharashtra (see Extended data) (Kochhar, 2022).\n\nGreen-building certification agencies are equipped to support local bodies for data capture, management, and public availability of data. Availability of updated and correct data on sustainability performance at a local level has traditionally been a challenge, which has been addressed in the case of PCMC to some extent. Building a robust system for demand-side resource-consumption data collection and analysis at local and state levels will contribute significantly toward collating information for India’s INDCs.\n\nThe use of existing green-building rating tools enables effective implementation of resource-efficiency policies at the building and municipal levels, enables municipal bodies to measure and monitor progress, and provides a feedback mechanism for modifications required (if any) in the rating systems.\n\nFor project proponents and municipal bodies, understanding the cost impact of green-rated buildings over a project’s lifecycle is crucial for designing appropriate incentive mechanisms. Further study on building performance and cost analysis of GRIHA-rated projects is required, which will also enable designing suitable incentives at state or municipal levels.\n\nThe recommendations for states and municipal bodies to institutionalise GRIHA and incentivise projects are as follows:\n\n\n\n• Develop relevant schemes including GRIHA, which should clearly highlight priorities and intentions by ensuring that GRIHA is embedded in an existing framework or issued via an official government order.\n\n• Adopt the Smart Cities Program in its entirety and incorporate the “at least 80% buildings (in redevelopment and greenfield) should be energy efficient and green buildings” criterion. States and municipalities (especially where SCM and AMRUT schemes are being actively implemented) should ensure resource efficiency through GRIHA adoption.\n\n• Mandate GRIHA adoption for government projects. For example, Maharashtra PWD had instructed all new buildings to be GRIHA compliant and upgraded 300 buildings in the state in 2019 according to GRIHA.\n\n\n\n• Financial or other incentives for developers and occupants should be structured with an objective to provide proportionate compensation for green-building-linked additional costs incurred by a project. The incentive packages should be targeted, consistent, and coordinated at all levels.\n\n• Set up a time frame and mechanism to convert policy incentives into mandatory regulations.\n\n• Support initial investments for green-building schemes, providing innovative financing instruments to projects. For example, increased investment funds may be made available for green-building developers at reduced interest rates from allied banks and other financial institutions.\n\n\n\n• Disseminate information in print and electronic media to enhance outreach. Ensure all information is made available through a regularly updated official website. Notices should be displayed on the main webpage and office bulletin boards. An internal mechanism for systematic website maintenance may be set up.\n\n• The incentive model should clearly mention the project cycle and stage(s) at which the linked incentives shall be released. The timelines and penalty clauses should be mentioned, and timely incentive release should be ensured.\n\n• Set up a dedicated Environment Cell and Review Committee comprising experts and/or a GRIHA official with clearly stated responsibilities. This expert network may act as a single point of contact for all green-building-related queries and liaison between various departments for releasing incentives.\n\n\n\n• Create awareness about the prolonged and long-term benefits of green construction by developing relevant resource material.\n\n• Invest in capacity-building programs for the public and the training of staff responsible for implementing green-building schemes. Ensure their inclusion in the annual budget and timeframe.\n\n• Maintain clarity and transparency in the incentives/processes offered/followed to ensure more uptake by citizens.\n\n• Conduct annual events and award local achievements in the green-building field.\n\n• Simultaneous efforts required in building markets for energy- and resource-efficient technologies, skills, and building materials.\n\n\n8. Conclusion\n\nThis paper examines select institutional mechanisms for incentivising green-rated projects. Project-level implementation mechanisms to propose recommendations that may be adopted by various municipal bodies were studied. Currently, only 25/100 smart-city proposals include GBRSs to achieve essential SCM goals. Based on feedback from certification agencies, project proponents, and government officials, information dissemination on institutional mechanisms for municipal bodies to integrate GBRSs within their existing framework is vital. GBRSs will ensure effective ground implementation of resource efficiency and support upcoming smart cities to fulfil CSCAF requirements.\n\nImplementation of the proposed recommendations provides municipal bodies a huge opportunity to strengthen their initiatives in the green-building domain and report CC mitigation, adaptation, and resilience measures to CSCAF using GRIHA parameters. These recommendations can also serve as useful guidelines for local-government agencies in other developing countries for implementing and encouraging green initiatives in the residential building sector for CC mitigation and adaptation.\n\nCSCAF, which incorporates GBRSs (including GRIHA) to assess municipal bodies’ performance, will also benefit and be able to report achievements of municipal bodies at national and international levels only when data are collated at the local level. Setting up eco-cells with the GRIHA Council’s support will facilitate data collection and reporting, thereby establishing a vertical connect while ensuring ground implementation.\n\nDevelopment authorities that are currently ineligible under SCM but are taking initiatives on green buildings are encouraged to express their interest in being included in SCM. Similarly, identified smart cities that have adopted several green-building measures are encouraged to report the same for effective and transparent implementation of national policies and commitment to CC mitigation, adaptation, and resilience.\n\nThis study had some limitations in, primarily in terms of data and information collection:\n\n(1) Project proponents are hesitant about sharing financial and building-performance data.\n\n(2) Access to municipality/local-body data is challenging as information in most cases is neither digitised nor easily accessible.\n\n(3) Information about GBRS-linked incentives in the remaining states/UTs is inaccessible; collecting such information physically was extremely difficult during 2019–2021 because of COVID-19-mandated travel restrictions.\n\nFurther study on building performance and cost analyses of GRIHA-rated projects is required to facilitate appropriate design of incentives according to local institutional mechanisms.\n\n\nData availability statement\n\nOpen Science Framework: 2020_0825_Green Building Policies and Initiatives and working document.\n\nhttps://doi.org/10.17605/OSF.IO/MX74U (Kochhar, 2022)\n\nThis project contains the following extended data:\n\n2020_0825_Green Building Policies and Initiatives_working document:\n\nList of States and UTs\n\nSmart Cities\n\nAnalysis\n\nSavings and cost_rating systems\n\nSCAF indicators\n\nHaryana incentives\n\nIncentive wise_Municipal level\n\nProjects-Rating data\n\nMinistry wise_Central level\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\nAuthor contributions\n\nPriyanka Kochhar: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Namrata Mahal: Data Curation, Formal Analysis, Writing – Review & Editing; Sanjay Seth: Resources, Supervision, Writing – Review & Editing; Mandeep Singh: Resources, Supervision, Writing – Review & Editing.", "appendix": "Acknowledgements\n\nWe are grateful to all local body officials for their valuable inputs and useful discussions. We thank Ms. Shibani Choudhary, Project Officer at GRIHA Council for providing technical support. We also thank all promoters and consultants for their inputs on the green residential projects.\n\n\nReferences\n\nAdministrative staff College of India and Natural Resources Defense Council: Greener Construction Saves Money: Incentives for Energy Efficient Buildings Across India.March 2014. Retrieved on October 27, 2021. Reference Source\n\nBureau of Energy Efficiency, Government of India: Report on Impact of Energy Efficiency Measures for the year 2018–19.March 2020. Retrieved on October 23, 2021. Reference Source\n\nBureau of Energy Efficiency: Buildings.2021. Reference Source\n\nBureau of Indian Standards: 2016. National Building Code.(Last updated on December 14, 2018). Reference Source\n\nCentral Public Works Department. Office Memorandum.March 16, 2009. Reference Source\n\nClimate Centre for Cities: Knowledge Repository. Retrieved on October 23, 2021. 2021. Reference Source\n\nColenbrander S, Gouldson A, Roy J, et al.: Can low-carbon urban development be pro-poor? The case of Kolkata, India. Environment and Urbanisation. 2016; 29(1): 139–158. Publisher Full Text\n\nCW Staff: Green footprint. Construction World2014 March 1.\n\nGRIHA Incentives: Retrieved on October 26, 2021. Reference Source\n\nGe M, Friedrich J, Vigna L: 4 Charts Explain Greenhouse Gas Emissions by Countries and Sectors.February 06, 2020. Retrieved on October 21, 2021. Reference Source\n\nGovernment of India: States and Union Territories.2021. Accessed on October 30, 2021. Reference Source\n\nGraham P, Rawal R: Achieving the 2°C goal: the potential of India’s building sector. Building Research and Information. 2019; 47(1): 108–122. Publisher Full Text\n\nGRIHA Council: Adaptation and climate resilience features in the GRIHA rating. New Delhi, Delhi, India.September 11, 2020.\n\nGRIHA Council: About Us (online).2021. Reference Source\n\nGRIHA Council: NOIDA and Greater NOIDA embrace GRIHA. GRIHA incentives.October 20, 2012. Accessed on October 30, 2021. Reference Source\n\nGRIHA Council: Incentive release approval for GRIHA rated project by PCMC. News & Updates.September 2019. Accessed on October 30, 2021. Reference Source\n\nIndia’s Intended Nationally Determined Contribution: Working Towards Climate Justice: 2021. Retrieved on October 21, 2021. Reference Source\n\nIndian Green Building Council: 2015. Government Incentives to IGBC-rated Green Building Projects.Retrieved on October 26, 2021 Reference Source\n\nInternational Energy Agency: 100 Smart Cities Mission.2021. Accessed on October 30, 2021. Reference Source\n\nKochhar P: Green Building Policies and Initiatives working document.2022, January 26. Publisher Full Text\n\nKochhar P: The ‘State of Play’ of sustainable buildings in India. Paris: United Nations Environmental Programme DTIE Sustainable Consumption & Production Branch.2010. Accessed on October 30, 2021. Reference Source\n\nKochhar P, Singh M: Case study of GRIHA rated institutional buildings Examining green building features and building energy systems to facilitate of GRIHA rated projects to facilitate design for all. Design for All Institute of India. 2021; 16(1): 61–80.\n\nMahal N: Formulating guidelines for effective implementation of Green Building Scheme of the Urban Local Bodies. Int. J. Eng. Res. Appl. 2016; 6(11): 19–27.\n\nMinistry of Environment and Forests, Government of India: Office Memorandum, No. 19-58/2011-IA.III.June 27, 2011.\n\nMinistry of Environment, Forests, and Climate Change, Government of India: The Gazette of India: Extraordinary.2014. Reference Source\n\nMinistry of Housing & Urban Affairs: Press Release.September 11, 2020. Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: SAAP 2017–2020. Accessed on October 30, 2021a. Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: SAAP - Atal Mission for Rejuvenation and Urban Transformation.2015. Retrieved on October 26, 2021. Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: ClimateSmart Cities.2019. Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: Thrust Areas.2021b. (online, last updated on October 25, 2021). Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: About Smart Cities Mission (online).2021c. Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: Assessment Framework Overview.2021d. Accessed on October 30, 2021. Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: Smart Cities.2021e. Accessed on October 30, 2021. Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: City Challenge.2021f. Accessed on October 30, 2021. Reference Source\n\nMinistry of Housing and Urban Affairs, Government of India: The Mission.June 2015b. Accessed on October 30, 2021. Reference Source\n\nMinistry of New and Renewable Energy, Government of India: 2017. Annual Report 2016–17.Reference Source\n\nMinistry of New and Renewable Energy, Government of India: Annual Report 2019–20.2020. Reference Source\n\nMinistry of New and Renewable Energy, Government of India: Annual Report 2018–19.2021; Page 70. Retrieved on October 22, 2021. Reference Source\n\nMinistry of New and Renewable Energy, Government of India. December 13: Lok Sabha Questions. Lok Sabha Question and Answer Portal [Online].2018. Reference Source\n\nMinistry of New and Renewable Energy, Government of India: March 2018. Thirty Second Report Standing Committee on Energy (2017–18). Reference Source\n\nMinistry of Non-conventional Energy Sources (Urban, Industrial, and Commercial Group), Government of India: Modified Scheme (Notification), (2009).2021. Retrieved on October 29, 2021. Reference Source\n\nMinistry of Statistics and Programme Implementation, Government of India: Energy Statistics 2019.March 2019. Retrieved on October 23, 2021. Reference Source\n\nMinistry of Urban Development, Government of India: Model Building Bye Laws.2016. Accessed on October 30, 2021. Reference Source\n\nMinistry of Urban Development, Government of India: Smart Cities Mission Statement and Guidelines.May 2015. Retrieved on October 26, 2021. Reference Source\n\nNational Institute of Urban Affairs: ClimateSmart Cities Assessment Framework 2.0 – Chapter 4: Indicator Description.2021. Retrieved on October 23, 2021. Reference Source\n\nNRI News: GRIHA Council organised “Paryavaran Rakshak programme for occupants of Devrai Phase-II, Pune.September 21, 2019. Accessed on November 1, 2021. Reference Source\n\nPimpri Chinchwad Municipal Corporation: Green Building Initiative.2021. Accessed on October 30, 2021. Reference Source\n\nRamkumar S: Why Green Ratings for Buildings Matter?.2020. Retrieved on October 22, 2021. Reference Source\n\nRight To Information query filed with the Town and Country Planning Organisation: reference no: TACPO/R/E/20/00261. Filed on September 1, 2020, obtained on September 4, 2020.\n\nSharma D, Tomar S: Mainstreaming climate change adaptation in Indian cities. Environment and Urbanization. 2010; 22(2): 451–465.\n\nSmith RM, Pathak P: Urban Sustainability in India: Green Buildings, AMRUT Yojana, and Smart Cities. Grant B, Liu C, Ye L, editors. Metropolitan Governance in Asia and the Pacific Rim. Singapore: Springer; 2018. Publisher Full Text\n\nTCP Haryana: Tentative EDC rate for different uses in various Urban Estates of Haryana calculated as per decision taken by the Council of Ministers in its meeting held on 03.02.2016.February 3, 2016. Reference Source\n\nThe Energy and Resources Institute: Project Report on ‘Review and Revision of CPWD Documents to Include Energy Efficiency Parameters and Capacity Building of Professionals’.2012. Reference Source\n\nThe Energy and Resources Institute: Energy Efficiency Potential in India.2018. Reference Source\n\nTown and Country Planning Department, Haryana Government: The Haryana Building Code 2017.2021. Accessed on October 30, 2021. Reference Source\n\nUN Environment and the International Energy Agency: Global Status Report 2017.2017. Reference Source\n\nUNFCC: The Paris Agreement.2021. Retrieved on October 28, 2021. Reference Source\n\nUnited Nations: 68% of the world population projected to live in urban areas by 2050, says UN\|UN Desa Department of Economic and Social Affairs. United Nations.May 16, 2018. Reference Source\n\nUttar Pradesh Real Estate Regulatory Authority: View Projects.September 14 2020. Accessed on November 1, 2021. Reference Source\n\nVK:e environmental: Complete, GRIHA, Projects, Pune, Residential. Devrai, Phase II.September 2019. Accessed on October 30, 2021. Reference Source\n\nWorld Green Building Council: New report: The building and construction sector can reach net zero carbon emissions by 2050.September 23, 2019. Reference Source\n\nWRI India: Greening Indian Cities Through Efficient Buildings: A guidebook for local governments to design and implement green buildings policies prepared by WRI India.2020. Retrieved on October 24, 2021. Reference Source\n\nWRI: What is an INDC?.2021. Retrieved on October 28, 2021. Reference Source\n\n\nFootnotes\n\n1 Premium is a fee paid by developers to PCMC, similar to infrastructure tax paid in other municipal corporations.\n\n2 74.28 Indian Rupee = 1 USD (as on January 8, 2022).\n\n3 According to discussion with Noida Authority officials, the compounding fee for projects in Greater Noida is same as that for projects in Noida." }	[ { "id": "122674", "date": "21 Feb 2022", "name": "Hina Zia", "expertise": [ "Reviewer Expertise Green buildings", "Climate Change and resilience", "Urban regeneration", "Ekistics", "Urban ecology", "economics of green buildings" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article attempts to critically examine and push forward the possibilities offered by a whole range of institutional mechanisms for incentivizing GBRS (with a focus on a specific rating system GRIHA) for catalysing climate-change mitigation and adaptation in India. The authors have touched upon a very important aspect of making the building sector 'green' and thus contribute to NDCs. The literature review and coverage of the variety of policy tools at local and state levels is comprehensive. There are however, few minor suggestions to further achieve the intended objectives.\nIt is suggested to improve the section on methods, specifically the part on details as to how many types and numbers of stakeholders from various geographies were selected for interviews. What specific inputs were intended to be received by interviewing these stakeholders? 'Structured questionnaire' is mentioned in the text but there is no further information available on the kind of questions asked, variety of stakeholders' interviewed, number of such interviews, etc.\n\nAn attempt to distinguish between advantages/challenges of policy tools such as mandatory codes and voluntary GBRS (including GRIHA) in Indian context (based on the experiences of various states/cities already covered by authors) would be very helpful.\n\nFurther deliberation on the key lessons learnt which has not been covered by any other studies (like WRI 2020 report) can help local government and state governments to take further necessary action. For instance, the authors have conducted structured interviews with few government entities and private developers. Insights from both supply and demand side of green buildings would add further value to this article.\n\nCSCAF 2.0 intends to scale the assessment tool from 100 smart cities to 500 cities. The authors may accordingly modify the article in the relevant section.\n\nAll Tables and Figures need to be properly 'sourced' as per any APA norms. Avoid repetitions, for instance, Table 2 has several repetitions.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] }, { "id": "126429", "date": "19 Apr 2022", "name": "Walaa S.E. Ismaeel", "expertise": [ "Reviewer Expertise Green building design and assessment" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper discusses the role of GRIHA green building rating system to catalyze climate change mitigation and adaptation. The topic is interesting, nevertheless, the paper is poorly prepared. It is rather a report than a scientific manuscript. The aim is not properly defined and it was not clear how the authors intended to address it quantitatively.\nThe abstract is rather generic. Revise the abstract to be concise, briefly summarizing the aim, objectives, method, results and conclusion of the study. Currently the abstract is long and some of the text can move to the introduction.\n\nKeywords: Revise keywords, they are too many\n\nThe method is not properly explained and it is not clear how did it get to the results.\n\nThe discussion and conclusion should be also revised to pinpoint the novelty of the study compared to other studies.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/11-153
https://f1000research.com/articles/11-147/v1	04 Feb 22	{ "type": "Review", "title": "Imaging the delayed complications of childhood Kawasaki disease", "authors": [ "Andrew Crean", "Lee Benson", "Ashish Shah", "Kelly Han", "John Lesser", "Brian W. McCrindle", "Lee Benson", "Ashish Shah", "Kelly Han", "John Lesser", "Brian W. McCrindle" ], "abstract": "This review will discuss the long-term complications of Kawasaki disease with a particular focus on imaging surveillance of the coronary arteries in adolescence and adult life. The relative advantages and disadvantages of each modality will be illustrated with practical examples, demonstrating that, in many cases, a multimodality imaging strategy may be required.", "keywords": [ "Kawasaki disease", "cardiovascular magnetic resonance", "CMR", "cardiac CT", "positron emission tomography", "PET", "intravascular ultrasound", "IVUS", "optical coherence tomography", "OCT" ], "content": "Introduction\n\nKawasaki disease (KD) is a systemic vasculitis of unknown etiology. Coronary arterial complications, both in acute or chronic phase of the illness, result in high morbidity and mortality.\n\nVascular remodeling characterized by fibroblastic proliferation and matrix metalloproteinase deposition results in progressive fibrosis and, eventually, stenosis.1 The flow pattern in an aneurysmal segment is characterized by recirculation, reduced wall shear stress and stasis, whereas there is flow acceleration through stenotic lesions.2 Such flow alterations result in platelet activation, which, in the presence of endothelial dysfunction, creates a highly thrombogenic milieu, and may result in thrombotic occlusion of the coronary arteries.3\n\nAlthough aneurysmal coronary artery dilatation is seen initially in pediatric acute KD, the subsequent complications of aneurysm thrombosis, distal embolism and infarction, or inter-aneurysm coronary artery stenosis, are more often seen much later in life. As a result, the sufferer of childhood KD will require life-long surveillance throughout adulthood in those cases where coronary artery dilatation fails to regress in the convalescent period. This review will describe these complications and offer suggestions to guide the selection of imaging modality (Table 1), through discussion of each in turn.\n\nEchocardiography is always the initial tool in the assessment of the patient with Kawasaki disease. Young children generally have excellent echocardiographic windows with the proximal portions of the coronary arteries being fully visualized. Imaging features to note include coronary artery dilatation (which on occasion may be subtle); ectasia and lack of distal tapering; frank aneurysmal formation; and perivascular echogenicity or “brightness” due to inflammation of the arterial wall (in acute presentations). Aneurysms may be isolated or segmental with intervening ‘skip areas’ of normal vessel and they may be either saccular or fusiform in shape. Proximal aneurysms can be accurately measured and evaluated for internal thrombosis or occlusion (Figure 1).\n\nA) Thrombus (asterisk) within the proximal portion of the large LAD aneurysm. B) Accelerating narrowed diastolic flow jet around the peripheral rim of the thrombus. LAD, left anterior descending.\n\nThe examination should also include a formal evaluation of cardiovascular function. Acutely unwell patients frequently have an associated myocarditis and may have wall motion abnormalities on 2D or 3D imaging. Associated pericarditis may present with pericardial effusion. Myocardial deformation tracking using speckle tracking may demonstrate improvement early in the recovery period, ahead of global measures such as ejection fraction,4 but may also remain abnormal in the long term, especially in more severely affected patients.5\n\nAcute coronary artery thrombosis, usually the result of in situ thrombosis within an aneurysm, may result in myocardial infarction or stunning and profound regional wall motion abnormality. Similarly, distal embolization of thrombus within an aneurysm may lead to myocardial infarction, wall motion abnormality and eventually regional thinning.\n\nValvular involvement in Kawasaki disease is reported but is rarely a prominent feature. Nonetheless the presence of severe mitral regurgitation should raise the question of underlying myocardial ischemia and secondary papillary muscle dysfunction.\n\n\nNuclear perfusion imaging\n\nNuclear medicine techniques were, for a long time, the mainstay of non-invasive imaging in Kawasaki disease for the detection of flow-limiting lesions and infarction (Figure 2). The ready availability of SPECT equipment together with the ability to combine perfusion and treadmill imaging has made this, historically, the most widely used approach to following patients in the chronic phase, both before and after coronary artery bypass surgery.6 There are no data in a sufficiently large sample from which to derive diagnostic accuracy estimates of stress SPECT; however, the ability of young people to exercise to high levels together with (generally) lower body mass index, and hence less photon attenuation, compared with the elderly atherosclerotic population, may lead to fewer non-diagnostic or false positive studies. Nonetheless, there are strong arguments for following younger patients by alternative means that do not expose them recurrently to ionizing radiation, such as stress echo and stress CMR.\n\nA) Nuclear SPECT perfusion study demonstrating a largely fixed infero-lateral defect on polar map. B,C) Late gadolinium enhancement reveals a non-transmural scar (white arrows) in the same area. D) Black-blood CMR shows circumflex aneurysms with thrombus (asterisks). E) Angiogram confirms Cx aneurysms with poor distal flow.\n\nNuclear cardiology has, however, provided insights into Kawasaki disease, particularly from positron emission tomographic (PET) imaging - which can provide higher accuracy for disease detection than SPECT perfusion imaging, and with lower radiation exposure.7,8 It has been known for a number of years that coronary endothelial function is abnormal following Kawasaki disease. One early paper examined coronary vasodilatory function in patients who had had Kawasaki disease but without any overt coronary involvement, and compared resting and stress flow to that in normal age-matched controls. Resting flow, measured using 13N-ammonia, was comparable in both groups, but interestingly the Kawasaki group had lower levels of hyperemic stress flow (and hence reduced flow reserve) despite ostensibly normal coronary arteries, possibly due to increased microvascular dysfunction.9 Hauser et al. demonstrated similar findings in children with normal epicardial arteries after the onset of Kawasaki disease.10 These observations were extended by Furuyama et al. who demonstrated similar findings, using 15O PET, in Kawasaki patients with regressed aneurysmal dilatation; but, using the cold pressor test to generate hyperemia, additionally showed impairment of the endothelial-dependent pathway.11 A more recent small study demonstrated even greater impairment of endothelial-dependent vasodilatation (as assessed by cold pressor test) in patients with frank aneurysmal dilation compared to those with regressed aneurysms, arguing for a gradation of endothelial damage across the spectrum of disease.12\n\nRubidium-82 generators have now been available for several years and this tracer might be an additional clinical tool to assess further the myocardial blood flow metrics on dynamic PET. There are, as yet, no reports of myocardial blood flow assessment using 82Rb in Kawasaki disease.\n\n\nCardiovascular magnetic resonance (CMR)\n\nCMR is arguably the single most useful non-invasive modality for following patients from the age of 8 years and upwards. The strength of CMR lies in the multiplicity of techniques available for assessing structure, function, and tissue characterization.\n\nStress perfusion MRI is increasingly being performed in congenital and inflammatory coronary conditions.13–15 The overall diagnostic accuracy has not been adequately established for non-atherosclerotic coronary conditions, but based on data from the CEMARC study is likely to be higher than for SPECT.16 Most centers currently interpret stress CMR images qualitatively (Figure 3). Interestingly, quantitative perfusion CMR has demonstrated abnormalities of microvascular function, which is entirely concordant with the available PET literature.16 Nonetheless, qualitative perfusion imaging in Kawasaki Disease can be problematic, as extensive collateral networks may form and, even in the setting of total occlusion, perfusion can appear qualitatively normal. It is for this reason that we usually combine cardiac CT and CMR in our practice (Figure 4).\n\nA,D) Calcific cast of RCA (arrow) is noted prior to contrast injection. B,C) Note that the contrast column does not touch the vessel edge outlined by the calcium, consistent with the presence of laminar thrombus. A focal tight mid RCA stenosis is present (arrow). E) Area of hypoperfusion (arrow) in the basal infero-septum on dipyridamole stress CMR. F) Whole heart coronary MRA of RCA corresponding to image in C – there is good correlation for aneurysm morphology, but note that the RCA distal stenosis is less well visualized due to inferior spatial resolution of CMR compared to conventional angiography.\n\nA,B) A zone of subendocardial hypoperfusion is evident in the RCA territory (arrows, top panels). Note the lack of underlying scar on the corresponding LGE images (bottom panels). C) Cardiac CTA shows a long segment of irregular and hypo-enhanced RCA suspicious for diffuse disease with high-grade stenoses. D) Coronary angiography in fact shows RCA occlusion with extensive bridging collaterals from proximal to distal RCA forming a ‘woven’ vessel. Further collateralization is seen from the Cx to the distal RCA (arrow, bottom panel). Extensive collateralization like this may reduce the size of the perfusion defect (as here) and the true ischemic burden can be difficult to judge. This young woman wished to get pregnant and went ahead without revascularization after Bruce protocol treadmill stress echo where she managed 13 METS of activity without symptoms, ECG changes or wall motion abnormality.\n\nIt should be recognized that inducible perfusion defects may be evident even after coronary artery bypass surgery, as incomplete revascularization is often inevitable.\n\nLate gadolinium enhancement (LGE) imaging provides clear images of scarred and infarcted segments due to prior coronary occlusion and/or embolism (Figure 5). The technique is recognized as being highly sensitive for even very small volumes of scar.\n\nA) Late gadolinium enhancement (LGE) acquired in 2010 demonstrates a small rim of thrombus (arrow) in the more distal of 2 sequential Cx aneurysms. B) Repeat in LGE in 2012 reveals increased thrombus burden in this aneurysm (white arrow) and new layers of thrombus in the more proximal aneurysm (black arrows). The patient had not been compliant with anticoagulation during this period. C & D) Mid ventricular short-axis and 2-chamber LGE sequences demonstrate partial thickness infarction (arrows) in the basal to mid inferior and inferolateral walls extending into the inferoseptal wall related to embolism into this dominant circumflex coronary artery.\n\nLess well-appreciated is the value of LGE imaging for detecting intra-coronary thrombus. Patients with giant aneurysms have altered rheology and altered wall characteristics; furthermore, abnormal shear forces may adversely affect platelets. Therefore all three components of Virchow’s triad are often present. Large occlusive thrombus is rarely a difficult diagnosis since the presentation is usually one of acute myocardial infarction. Smaller amounts of thrombus can be quite challenging to visualize and are often missed by echo, as they usually line the walls of an aneurysm without causing much reduction in internal diameter or disrupting flow. The LGE technique provides excellent visualization of ventricular thrombi due to the low signal returned from thrombus contrasting well with the grey/white signal from the coronary blood pool (Figure 5).\n\n\nCardiac CT\n\nCardiac CT is a useful and reasonable method for ‘staging’ new referrals to the adult Kawasaki clinic. In our clinic it is a baseline test to establish the presence and extent of aneurysmal disease and stenosis and may act as a gatekeeper to subsequent coronary angiography.\n\nThe major advantages of cardiac CT are its rapidity, spatial resolution, and sensitivity for calcium. Modern scanners can complete acquisition of the target anatomy in a few seconds (or less) making CT very suitable for the restless or claustrophobic. Spatial resolution is in the region of 0.5 mm for most scanners which is adequate for the depiction of aneurysms, thrombus and normal or ectatic coronary segments. Clinically useful coronary coverage is almost invariably greater by CT than it is by echo17 and good correlation with conventional coronary angiography has been demonstrated18 (Figure 6).\n\nA-C) Conventional angiographic views display 2 giant aneurysms in the RCA. D,E) Curved multiplanar reformats from cardiac CT data set demonstrate good correlation and more clearly depict wall calcification and lack of mural thrombus. F) Volume-rendered image from a cardiac CT data set represents an alternative visual method for displaying CT findings.\n\nUnlike CMR, cardiac CT is exquisitely sensitive for even small amounts of calcium. Calcium seems to be mainly associated with aneurysms and in patients above the age of ten.19 It is uncertain whether this matters, as it remains unclear whether Kawasaki patients have increased propensity to develop accelerated atherosclerotic lesions as they get older. Occasionally, however, calcification may occur at the site of minimal coronary dilatation, presumably secondary to healing following immune-mediated vasculitis (Figure 7).\n\nA) Initial coronary CT demonstrates subtle soft plaque/vessel wall thickening (white asterisks) in the proximal LAD. B) Calcium score, 3 years later, now shows new overlying calcification (black asterisks) implying mineralization secondary to chronic low-grade inflammation. Statin therapy should be considered for all KD patients although the level of evidence for treatment is weak.\n\nCardiac CT may also be used to identify intra-aneurysmal thrombus.20 A delayed phase data set acquired roughly 60 seconds after injection is required in addition to the arterial phase coronary acquisition, to permit full mixing of iodine with blood, which may be delayed in larger aneurysms, leading to filling defects and pseudo-thrombus (Figure 10).\n\nMyocardial scar, due to infarction or embolism, may also be recognized when late iodine enhancement imaging is performed with care. This is, however, an area where CMR retains superiority.\n\nFinally vasodilator stress CT has demonstrated incremental benefit in identification of flow-limiting disease in adult atherosclerotic disease.21 There are no published data on this technique in Kawasaki disease, though it could be attractive in theory due to benefits derived from perfectly co-registered anatomy and perfusion data sets (Figure 8).\n\nA-C) Basal, mid and apical short axis reconstructions of a stress CT data set demonstrating an LAD territory perfusion defect in a Kawasaki patient with LAD aneurysm and prior occlusion.\n\n\nCardiac catheterization\n\nAngiography remains the tool of choice in symptomatic patients or those with equivocal non-invasive imaging. As a ‘real-time’ injection of contrast is made, the degree of sluggish or swirling flow is much more obvious than with cross-sectional techniques (Figure 9). Nonetheless, we have occasionally seen cases where swirling flow was misinterpreted as ball valve thrombus in a proximal giant aneurysm (Figure 10).\n\nMultiple frames are shown from the left coronary injection of a conventional angiogram. A large saccular proximal LAD aneurysm is outlined. Note the extreme delay before contrast fully opacifies the distal LAD at frame 150 from the start of injection. It is this intra-coronary stasis that accounts for the high thrombotic risk in this patient population, even on anticoagulants.\n\nA) Right coronary injection demonstrates a large proximal aneurysm with an apparent central oval lucency (white asterisk). This was interpreted as a ball-valve thrombus in this young man with chest pain symptoms. B) Same-day coronary CT acquired 1 minute after injection shows completely uniform iodine intensity within the aneurysm (black asterisk), confirming the absence of any real thrombus – indicating that the catheter finding was the result of swirling flow and incomplete mixing of blood and iodine immediately after injection.\n\nThrombotic occlusion of the coronary artery is a feared complication in patients with KD, not only in the acute phase of illness, but also many years later. In a case series of 50 adult patients, thrombotic occlusion of the aneurysmal segment was the most common complication. The majority of the patients were treated with balloon angioplasty and/or thrombolytic use; rotational atherectomy and stenting were used in only one patient each. IVUS examination in some of these patients demonstrated thickened intima, as expected, but no atherosclerotic changes were noted.22\n\nBalloon angioplasty, even with high-pressure balloons, is unlikely to be successful in adult patients, due to presence of dense fibrosis.23 Such an approach can result in neo-aneurysm formation that has the potential to result in stent malapposition. Similarly, because of variation in vessel caliber, due to interspersed aneurysm and stenotic lesions, achieving satisfactory stent apposition can be challenging,24 and malapposed stent struts are an independent risk factor for stent thrombosis.\n\nPercutaneous coronary intervention in patients with KD should be guided by intra-coronary imaging that can help differentiate between densely calcified lesions versus fibrotic ones, as rotational atherectomy can be successfully used to treat the former, whereas use of a cutting balloon would be preferred in the latter.25\n\nVisual assessment of coronary angiography has inherent limitations. FFR measurement has been validated and extensively used to assess physiologic significance of obstructive coronary artery disease. The presence of sequential narrowings in the same artery results in a cumulative impact on myocardial perfusion. Interestingly, not only coronary stenosis, but also aneurysmal dilatation results in ‘pressure loss’ due to turbulence, which may contribute towards worsening of coronary perfusion.26,27 In patients with KD, whose coronary arteries demonstrate diffuse stenosis and aneurysmal dilatation, visual assessment by angiography alone is inadequate, and FFR should be considered. One caveat though is that FFR assessment relies on vascular endothelial response to variety of vasoactive substances. Patients with KD are known to have endothelial dysfunction that can affect peak hyperemia, although the impact of such dysfunction on FFR measurement is not known.\n\nIntravascular ultrasound (IVUS) uses piezo-electric transducer or electronic phased array to sends out sound signals that are reflected by the tissue it passes through, according to their acoustic properties. IVUS examination of the coronary arteries, many years after an acute episode of KD often demonstrates thickened intima-media complex, and diffuse calcification at the sites of persisting or regressed aneurysms, but atherosclerotic changes are not usually seen.28\n\nOptical coherence tomography (OCT) measures backscatter of the light (optical echos), and offers the highest possible resolution images below 10 μm (Figure 11). OCT examination allows higher definition image acquisition of a fibrous cap (thickness, cellular infiltration and erosion), necrotic core, calcified lesions, plaque rupture, dissection plane, and stent apposition.29,30 These images are much clearer than the ones acquired with IVUS. OCT examination provides detailed tissue information, comparable with the histopathological examination of the coronary arteries.31 However, higher resolution comes at a cost of limited penetration; OCT may not be an imaging modality of choice in large or giant aneurysms.\n\n(A & E) Coronary angiogram demonstrating mild atherosclerotic disease. (B & F) OCT examination of these coronary arteries demonstrating normal intima-media where * demonstrates muscular media. (C & G) Angiography demonstrating coronary arteries many years after Kawasaki disease. OCT examination of these arteries demonstrates destroyed intima-media structure that is replaced by thick fibrosis (D) and disintegrated muscular layer (H). (I) Longitudinal reformatted OCT image of the same coronary artery demonstrated aneurysmal and stenotic segments along its length.\n\nNewly referred adults - with a history of Kawasaki disease in childhood - are staged with up to 3 levels of investigation. Level 1: Coronary CTA is performed first. If there is no evidence of coronary involvement (or no worse than only mild dilatation), no further investigation is required. If there is evidence of aneurysm or stenosis further testing is required. Level 2: The precise test chosen should depend upon availability and local expertise. If embolic infarction from aneurysmal thrombus is suspected, then CMR is often the most revealing test. Where the selected level 2 test is equivocal, we often proceed to an alternative level 2 test for confirmation. When there remains residual doubt, or if a level 2 test is positive, we proceed to the next step. Level 3: Invasive coronary angiography with or without hemodynamic assessment of severity of stenosis by FFR. While OCT and IVUS may be useful to assess stent deployment, percutaneous coronary intervention is often a poorer choice than bypass surgery for the majority of Kawasaki patients, given the variability in cross-sectional diameter of involved segments.\n\n\nConclusions\n\nKawasaki disease patients with known coronary involvement should be followed carefully throughout adult life. A single approach is unlikely to be able to reliably demonstrate all of the possible coronary and cardiac complications that can result from this disease. Appropriate use of imaging allows for early detection of complications with subsequent surveillance and may reduce subsequent morbidity and mortality.", "appendix": "References\n\nNewburger JW, Takahashi M, Gerber MA, et al.: Diagnosis, treatment, and long-term management of Kawasaki disease: a statement for health professionals from the Committee on Rheumatic Fever, Endocarditis and Kawasaki Disease, Council on Cardiovascular Disease in the Young. Circulation. 2004 Oct 26; 110(17): 2747–2771. PubMed Abstract \| Publisher Full Text\n\nSengupta D, Kahn AM, Burns JC, et al.: Image-based modeling of hemodynamics in coronary artery aneurysms caused by Kawasaki disease. Biomech Model Mechanobiol. 2012 Jul; 11(6): 915–932. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhillon R, Clarkson P, Donald AE, et al.: Endothelial dysfunction late after Kawasaki disease. Circulation. 1996 Nov 1; 94(9): 2103–2106. PubMed Abstract \| Publisher Full Text\n\nMcCandless RT, Minich LL, Wilkinson SE, et al.: Myocardial strain and strain rate in Kawasaki disease. Eur Heart J Cardiovasc Imaging. 2013 Nov; 14(11): 1061–1068. PubMed Abstract \| Publisher Full Text\n\nYu W, Wong SJ, Cheung Y: Left ventricular mechanics in adolescents and young adults with a history of kawasaki disease: analysis by three-dimensional speckle tracking echocardiography. Echocardiography. 2014 Apr; 31(4): 483–491. PubMed Abstract \| Publisher Full Text\n\nMostafa MS, Sayed AO, Al Said YM: Assessment of coronary ischaemia by myocardial perfusion dipyridamole stress technetium-99 m tetrofosmin, single-photon emission computed tomography, and coronary angiography in children with Kawasaki disease: pre- and post-coronary bypass grafting. Cardiol Young. 2015 Jun; 25(5): 927–934. PubMed Abstract \| Publisher Full Text\n\nMc Ardle BA, Dowsley TF, deKemp RA, et al.: Does rubidium-82 PET have superior accuracy to SPECT perfusion imaging for the diagnosis of obstructive coronary disease?: A systematic review and meta-analysis. J Am Coll Cardiol. 2012 Oct 30; 60(18): 1828–1837. PubMed Abstract \| Publisher Full Text\n\nParker MW, Iskandar A, Limone B, et al.: Diagnostic accuracy of cardiac positron emission tomography versus single photon emission computed tomography for coronary artery disease: a bivariate meta-analysis. Circ Cardiovasc Imaging. 2012 Nov; 5(6): 700–707. PubMed Abstract \| Publisher Full Text\n\nMuzik O, Paridon SM, Singh TP, et al.: Quantification of myocardial blood flow and flow reserve in children with a history of kawasaki disease and normal coronary arteries using positron emission tomography. J Am Coll Cardiol . 1996; 28: 757–762. Publisher Full Text\n\nHauser M, Bengel F, Kuehn A, et al.: Myocardial blood flow and coronary flow reserve in children with “normal” epicardial coronary arteries after the onset of Kawasaki disease assessed by positron emission tomography. Pediatr Cardiol. 2004 Apr; 25(2): 108–112. PubMed Abstract \| Publisher Full Text\n\nFuruyama H, Odagawa Y, Katoh C, et al.: Assessment of coronary function in children with a history of Kawasaki disease using (15)O-water positron emission tomography. Circulation. 2002 Jun 18; 105(24): 2878–2884. PubMed Abstract \| Publisher Full Text\n\nCicala S, Pellegrino T, Storto G, et al.: Noninvasive quantification of coronary endothelial function by SPECT imaging in children with a history of Kawasaki disease. Eur J Nucl Med Mol Imaging. 2010 Dec; 37(12): 2249–2255. PubMed Abstract \| Publisher Full Text\n\nSpiliotopoulos K, Yanagawa B, Crean A, et al.: Surgical management of a left anterior descending pseudoaneurysm related to Behcet’s disease. Ann Thorac Surg. 2011 Mar; 91(3): 912–914. PubMed Abstract \| Publisher Full Text\n\nTobler D, Motwani M, Wald RM, et al.: Evaluation of a comprehensive cardiovascular magnetic resonance protocol in young adults late after the arterial switch operation for d-transposition of the great arteries. J Cardiovasc Magn Reson. 2014 Dec 11; 16: 98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeva DP, Torres FS, Wald RM, et al.: The value of stress perfusion cardiovascular magnetic resonance imaging for patients referred from the adult congenital heart disease clinic: 5-year experience at the Toronto General Hospital. Cardiol Young. 2014 Oct; 24(5): 822–830. PubMed Abstract \| Publisher Full Text\n\nGreenwood JP, Maredia N, Younger JF, et al.: Cardiovascular magnetic resonance and single-photon emission computed tomography for diagnosis of coronary heart disease (CE-MARC): a prospective trial. Lancet. 2012 Feb 4; 379(9814): 453–460. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu Y, Sun K, Wang R, et al.: Comparison study of echocardiography and dual-source CT in diagnosis of coronary artery aneurysm due to Kawasaki disease: coronary artery disease. Echocardiography. 2011 Oct; 28(9): 1025–1034. PubMed Abstract \| Publisher Full Text\n\nCarbone I, Cannata D, Algeri E, et al.: Adolescent Kawasaki disease: usefulness of 64-slice CT coronary angiography for follow-up investigation. Pediatr Radiol. 2011 Sep; 41(9): 1165–1173. PubMed Abstract \| Publisher Full Text\n\nKahn AM, Budoff MJ, Daniels LB, et al.: Calcium scoring in patients with a history of Kawasaki disease. JACC Cardiovasc Imaging. 2012 Mar; 5(3): 264–272. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Agustin JA, Gomez de Diego JJ, Rodrigo JL, et al.: Right coronary artery aneurysm due to Kawasaki disease: A comprehensive assessment by multislice computed tomography. Int J Cardiol. 2014 May; 173(2): e12–e13. PubMed Abstract \| Publisher Full Text\n\nRochitte CE, George RT, Chen MY, et al.: Computed tomography angiography and perfusion to assess coronary artery stenosis causing perfusion defects by single photon emission computed tomography: the CORE320 study. Eur Heart J. 2014 May; 35(17): 1120–1130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsuda E, Abe T, Tamaki W: Acute coronary syndrome in adult patients with coronary artery lesions caused by Kawasaki disease: review of case reports. Cardiol Young. 2011 Feb; 21(1): 74–82. PubMed Abstract \| Publisher Full Text\n\nSugimura T, Yokoi H, Sato N, et al.: Interventional treatment for children with severe coronary artery stenosis with calcification after long-term Kawasaki disease. Circulation. 1997 Dec 2; 96(11): 3928–3933. PubMed Abstract \| Publisher Full Text\n\nShah AH, Abdel-Hadi H, Overgaard CB, et al.: Kawasaki disease and coronary intervention: A word of caution. Int J Cardiol. 2015 Dec; 201: 646–647. PubMed Abstract \| Publisher Full Text\n\nIto S, Suzuki T, Suzuki T: Adjunctive use of cutting balloon after rotational atherectomy in a young adult with probable kawasaki disease. J Invasive Cardiol. 2003. PubMed Abstract\n\nMurakami T, Tanaka N: The physiological significance of coronary aneurysms in Kawasaki disease.EuroIntervention; 2011; 944–947. Publisher Full Text\n\nYoshikawa T, Suma K, Sugawara M: Hemodynamic effects of bead-shaped aneurysm in Kawasaki’s disease (author's transl). Kokyu to junkan Respiration . 1980.\n\nSuzuki A, Yamagishi M, Kimura K: Functional behavior and morphology of the coronary artery wall in patients with Kawasaki disease assessed by intravascular ultrasound. J Am Coll Cardiol . 1996; 27: 291–296. PubMed Abstract \| Publisher Full Text\n\nFinn AV, Chandrashekhar Y, Narula J: IVUS and OCT: either or survivor … 2011.\n\nSuter MJ, Nadkarni SK, Weisz G, et al.: Intravascular optical imaging technology for investigating the coronary artery. JACC Cardiovasc Imaging. 2011 Sep 1; 4(9): 1022–1039. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBezerra HG, Costa MA, Guagliumi G, et al.: Intracoronary optical coherence tomography: a comprehensive review clinical and research applications. JACC Cardiovasc Interv. 2009 Nov; 2(11): 1035–1046. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "136684", "date": "10 Jun 2022", "name": "Diana van Stijn", "expertise": [ "Reviewer Expertise Kawasaki disease with focus on cardiovascular pathology and 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\nAndrew et al. have submitted \"Imaging the delayed complications of childhood Kawasaki disease\". The aim of this paper is to discuss imaging modalities used in Kawasaki disease patients during adolescence and adult life. First of all we would like to appraise the enormous effort in performing this hands-on review. Knowledge on cardiovascular imaging in adults and young adults with a history of Kawasaki disease is of great importance.\nMinor comments:\nOverall The authors may consider giving a bit more in-depth background information on the risks that come hand-in-hand with developing CAA after Kawasaki disease (mostly in patients with persistent giant aneurysms). Moreover, it would be nice to add some information on size-dependent long-term risk of having a small, medium and giant CAA. Also, few references are used throughout the article. Although the article discusses very useful expert opinions, it would be nice to add some references with supporting data (from this or other research groups) as well.\n1. Abstract We would strongly recommend using the term \"imaging modalities\" instead of \"imaging surveillance\". As imaging surveillance might suggest a hands-on flowchart of the desired imaging over time during follow-up. While the strength of this review lies more in depicting the potential of visualizing pathology with specific imaging modalities.\n2. Introduction “Coronary arterial complications, both in acute or chronic phase of the illness, result in high morbidity and mortality.” This sentence might give the impression that Kawasaki disease has a high morbidity and mortality. Please consider rephrasing this sentence and add absolute numbers and references with the risk of developing persistent CAA and consequent complications (such as thrombosis, myocardial infarction etc.).\n3. Introduction section \"echocardiography\" The authors mention \"perivascular echogenicity or \"brightness\"\" as important imaging features on echocardiography. Despite that early reports considered this as an early sign in KD 1, recent papers showed this to be a non-specific finding that can also be found in children without KD 2,3. Do the authors consider perivascular brightness an important hallmark in the (young) adult population, or is it as questionable as it is during early childhood?\n“Echocardiography is always the initial tool in the assessment of the patient with Kawasaki disease. Young children generally have excellent echocardiographic windows with the proximal sections of the coronary arteries being fully visualized.” Add references indicating that we don’t miss proximal CAA. The authors might also consider discussing whether (or how often) distal CAA may be missed by echocardiography?\n“Valvular involvement in Kawasaki disease is reported but is rarely a prominent feature. Nonetheless the presence of severe mitral regurgitation should raise the question of underlying myocardial ischemia and secondary papillary muscle dysfunction.” Please add references.\n4. CMR “Nonetheless, qualitative perfusion imaging in Kawasaki Disease can be problematic” A typo: Kawasaki Disease not Kawasaki disease. In the last few paragraphs many statements are made without references, please add references.\n5. Cardiac CT The authors mention something very important for the transition to adult care, namely \"staging\" new referrals by performing a CT. Would the authors, based on their expert opinion, advise pediatricians/pediatric cardiologists prior to referring a young adult to adult care, to perform a CT?\n“Cardiac CT is a useful and reasonable method for ‘staging’ new referrals to the adult Kawasaki clinic. In our clinic it is a baseline test to establish the presence and extent of aneurysmal disease and stenosis and may act as a gatekeeper to subsequent coronary angiography.” When do authors recommend using cardiac CT in the pediatric population?\nPlease consider discussing radiation exposure as a disadvantage of CT as well.\nSeveral statements are made without references, for example: “The major advantages of cardiac CT are its rapidity, spatial resolution, and sensitivity for calcium. Modern scanners can complete acquisition of the target anatomy in a few seconds (or less) making CT very suitable for the restless or claustrophobic. Spatial resolution is in the region of 0.5 mm for most scanners which is adequate for the depiction of aneurysms, thrombus and normal or ecstatic coronary segments.”\n4. Figure 12 This figure is of great value, knowing the scope of this article is more towards the value of imaging modalities and not so much surveillance. Could you still elaborate on how often you recommend to perform level 3 imaging? In children we tend to refrain from invasive coronary artery angiography and solely use it when intervention is needed. Therefore we perform follow-up mainly with level 1&2 in KD patients (with coronary artery involvement).\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": "154822", "date": "23 Dec 2022", "name": "Kai Sheng Hsieh", "expertise": [ "Reviewer Expertise Pediatric Cardiology", "echocardiography", "Pediatric cardiac imaging" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 discussed the imaging modalities in the late phase of childhood Kawasaki disease. The authors thoroughly reviewed the role of echocardiography, nuclear perfusion imaging, positron emission tomography, computed tomography, cardiovascular magnetic resonance angiography, coronary artery angiography and intervention, coronary fractional flow low reserve, intravascular ultrasound, and optical coherence tomography (SPECT/PET/CT/CMRAngiography/CAG/CFFR/IVUS/OCT). The review is comprehensive and informative. The quality of various figures also are good. The authors also list a table to display the relative strengths and weaknesses of commonly used imaging modalities for Kawasaki disease. The content of this manuscript is thus of sufficient quality and I will suggest to index this manuscript as its current form and no revision is necessary.\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": "163155", "date": "23 Mar 2023", "name": "Rakesh Kumar Pilania", "expertise": [ "Reviewer Expertise Kawasaki 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\nCrean et al. have submitted their review of imaging the delayed complications of childhood KD. Authors have provided comprehensive review on the subject. However, I have few comments.\n\nEcho:\nAuthor should define difference between dilatation, ectasia, and aneurysms clearly for benefit of readers. Authors can add images of echocardiography differentiating among these terminologies.\n\nThough ECHO may be an initial modality of imaging at presentation, however as the child grows ECHO may be challenging due to smaller acoustic window, inability to demonstrate the CAAs in mid and distal segments and LCx. complications like thrombus. stenosis or mural calcifications.\n\nEctasia is confusing terminology and nowadays not very common in use.\n\nLimitations of Echo need to be talked upon, especially identification of distal aneurysms, thrombosis, stenosis, calcifications.\n\nNuclear scan: Role of nuclear imaging is need to be balanced in current era where CTCA, ECHO and CMR would be more important imaging.\n\nCMR: CMR is most useful imaging modality in the late phase of disease. Authors have detailed almost very aspect of that. However, Article is silent on MRCA. Role of MRCA needs more literature search and its role and comparison with CTCA.\n\nCardiac CT:\n\nCTCA is definitely an imaging of choice for evaluation in an adult with history of KD.\n\nIt would be important to highlight the strengths of CTCA in comparison to 2D-Echo in identification of distal coronary artery involvement, entire course of coronaries, LCX, complications, stenosis etc. Singhal M et al. Distal coronary artery abnormalities in Kawasaki disease: experience on CT coronary angiography in 176 children1.\n\nRadiation exposure optimization protocol.\n\nAuthors describe CTCA as gate keeper modality: to be elaborated further\n\nDetection of calcification need to be more highlighted. Chakraborty R, et al. Coronary arterial abnormalities detected in children over 10 years following initial Kawasaki disease using cardiac computed tomography2.\n\nCardiac cath: authors have identified very specific indications for cath angio in a given patient with KD.\n\nAuthors have provided imaging approach in a complicated patient with KD. This figure is not cited in text. It would be better to expand on that before conclusions section with separate subheading. Authors have given equal weightage to rubidium stress PET to stress perfusion CMR and treadmill stress ECHO. As rubidium stress PET would have associated exposure with radiation exposure. Author can justify their approach.\n\nMinor errors: Author can use consistently use abbreviation for Kawasaki disease as KD throughout the manuscript. Please check spacing and abbreviations throughout the manuscript.\n\n‘\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-147
https://f1000research.com/articles/11-146/v1	04 Feb 22	{ "type": "Research Article", "title": "Knowledge and prevention practice against dengue vectors among dengue patients and general people in Chattogram, Bangladesh", "authors": [ "Sahidur Rahman", "Fatema Mehejabin", "Rumana Rashid", "Fatema Mehejabin", "Rumana Rashid" ], "abstract": "Background: Due to the absence of an effective vaccine for dengue, community-led vector control strategy could be a sustainable approach for dengue prevention. Therefore, this study aimed to assess people’s knowledge of dengue vectors and the practice of preventive measures to avoid vector mosquitoes by means of a structured questionnaire. Methods: A telephone-based survey was conducted between July 2019 to December 2019 from confirmed dengue patients and general people without dengue fever living in Chattogram, Bangladesh. Patients’ contact information was collected from two tertiary care hospitals. The level of knowledge and preventive practice were determined through the scoring of each participant against their responses. The association of individuals’ knowledge and practice scores with demographic variables was measured through chi-square and binary logistic analyses.\n\nResults: Overall, 61.9% of participants (72% case and 51.7% non-case) had good knowledge, whereas only 10.6% of them (12.7% case and 8.7% non-case) strongly practiced the mosquito prevention methods. However, significant variation in the level of knowledge was found between the two groups. Urban residents had 2.20 times higher knowledge compared to semi urban. Students and government officials had 3.39 times and 3.17 times better knowledge than general workers respectively. Permanent residents had 2.01 times better knowledge in comparison to the people living in temporary housing. In terms of mosquito preventive measures, semi-urban people showed 3.19 times (CI=0.97-10.52) stronger practice compared to rural people. Conclusions: This study suggests that dengue control strategies should focus on the effective practice of mosquito prevention by engaging community people.", "keywords": [ "Aedes mosquito", "dengue fever", "vector-borne disease", "knowledge and practice", "vector control", "dengue prevention" ], "content": "Introduction\n\nDengue fever is one of the most common vector-borne infectious diseases, endemic throughout the tropics and subtropics of the world. It is an acute mosquito borne illness and produces flu- like symptoms in humans after a short incubation period of 5-8 days (Du et al., 2021). Dengue virus is a single-stranded RNA virus under the genus Flavivirus possessing four unique serotypes DENV 1, 2, 3, and 4. However a new serotype was discovered in Malaysia which also has the potential to affect humans (Freeman et al., 2019). The virus is circulated through human-mosquito-human cycles and spreads through the bites of Aedes aegypti and Aedes albopictus mosquitoes. In addition, it may also spread from human to human without a carrier, in particularly through blood transmission, transplantation and needle puncture injuries (Wilder-Smith et al., 2019). Due to the common symptoms of dengue infection including high fever, rash, severe headache, joints, and muscle pain, it has also been known as “break-bone fever” (Abbasi et al., 2016). Sometimes, dengue fever can progress to the severe stage known as dengue hemorrhagic fever and manifest with blood in the urine or stool, bleeding gums, or bleeding from nose, indicating a critical condition of a patient.\n\nDengue fever has become one of the major public health concerns with a huge global burden in recent years. It has been representing a threat for around four billion inhabitants of more than 141 countries (World Mosquito Program, 2021). According to recent data, almost 100 million people show clinical manifestations every year with nearly 20,000 deaths (WHO, 2020). The 3/4th of the total burden was reported to be concentrated in the South East Asia and Western Pacific regions. A recent study has reported that about 52% of Southeast Asian people were found to be at risk of contracting dengue fever (Hossain et al., 2021). In Bangladesh, the first dengue case was detected in 1964. However, in 2000, the outbreak turned into a catastrophic situation, with 93 deaths among the 5,551 dengue cases in Dhaka, Khulna, and Chittagong city (Mutsuddy et al., 2019). In the following year, a remarkable reduction was observed in 2014 with 375 dengue cases, but the dengue fever epidemic has increased recently from 2,769 cases in 2017 to 10,148 cases in 2018 (Abir et al., 2021). In 2019, the incidence of dengue fever rose sharply, with 164 deaths from more than 100 thousand confirmed cases, representing an 11-fold increase in the incidence of dengue compared to previous year (Directorate General of Health Services (DGHS), 2020; Rafi et al., 2020).\n\nDifferent biological and socio-cultural factors have been found responsible for the spread of dengue infection in an area. The literature counts various factors including age, blood group, female sex, genetic polymorphism, co-morbidities, urbanization, stagnant water, geographic location, among others (Liu et al., 2018). A case-control study in study area (Chattogram city, Bangladesh) also revealed that traveling, house structure, day-time sleep, and house plants are potential risk factors for dengue infection of the residents (Rahman et al., 2021a). On the other hand, season and climate parameters like temperature, humidity, and rainfall also play an indirect role on dengue transmission. However, Aedes mosquitoes have a unique affinity to certain types of containers for breeding. Previous study found that discarded tires, plastic buckets, plastic drums, and coconut shells around houses were preferred for Aedes larvae production. Moreover, 17.35% of household in the study area were infested with Aedes mosquito larvae or pupae (Rahman et al., 2021b).\n\nThe lack of knowledge regarding dengue infection has been considered as one of the main problems in dengue epidemiology (Lian et al., 2006). According to a study conducted in Malaysia, a lack of awareness about dengue transmission and prevention strategies can enhance the risk of infection (Nguyen et al., 2019). A previous study in Malaysia revealed that people who have more knowledge about dengue are more likely to pursue more suitable dengue prevention measures compare to people with less knowledge (ROSLAN et al., 2020). Factors based on the good awareness and knowledge of dengue prevention measures are also responsible for drastic reductions in dengue transmission (Abir et al., 2021). Thus, evaluating people’s knowledge, attitude, and practice is of great importance for strengthening integrated control measures, which remain understudied. As there has been no effective vaccine approved so far, vector control and preventing mosquito bites through community empowerment and engagement is an effective option for prevention (Rahman et al., 2021b). To ensure a sustainable dengue prevention, increasing people’s knowledge and awareness towards vector control is highly important. Previous knowledge, attitude, and practice (KAP) studies mainly focused on clinical aspects of dengue fever while the vector perspectives remained neglected. Therefore, in this study we focused on mosquito vectors and assessed the general knowledge of people on dengue vectors and their practices regarding the prevention of mosquito presence in the Chattogram district, Bangladesh. Moreover, we compared the level of knowledge and prevention practice between dengue-infected and non-infected people. The findings of this study could serve as baseline data for researcher and policymakers.\n\n\nMethods\n\nThis cross-sectional study concerning knowledge, attitude and practices related to dengue prevention among the people in Chattogram, Bangladesh was conducted between July 2019 and December 2019. Our target participants were divided into two groups: recent dengue cases and apparently healthy people without dengue infection. We collected a list of confirmed dengue cases with contact details from the record of dengue ward of Chattogram Medical College Hospital and Bangladesh Institute of Tropical and Infectious Diseases (BITID). Both of these are the government tertiary care hospitals in Chattogram city. We included only those who were residents of the Chattogram district and got infected within the previous 15 days. Contact information of non-case participants were collected from the people who visited the outpatient department of the same hospitals. A comprehensive review of the literature was conducted to identify the factors determining the level of knowledge and dengue prevention practices among the people. All research participants or their guardians (for children) were assured that the data would be kept anonymous and use for research purposes only. Each participant had the right to leave the survey process of this study at any time. Participation in this study was fully voluntary and proceeding to the main parts of the interview happened upon verbal informed consent. No incentives were given to any respondent. We contacted 184 dengue patients and 200 non-infected persons. A few mobile phone numbers were unresponsive or the number holder was the patient’s relative. Moreover, partial data of participants who left without completing the survey were excluded from the analysis. Finally, we obtained complete data from 300 participants from a total of 384 contacted people with a response rate of 78.1%.\n\nData were collected within a two-month period, October and November 2019. We developed a survey questionnaire by reviewing literature and consulting experienced clinicians and epidemiologists. Data was collected through telephone-based interview. Most of the questions were multiple-choice checkbox types with Yes and No answers as well as ‘other’ options included with a few questions.\n\nThe aim and objectives of the study were clearly explained at the beginning of the survey. The questionnaire was divided into three parts. Part 1 comprised questions relating to socio-demographic details of the participants. Part 2 included questions on general knowledge of dengue and its vectors while Part 3 inquired on their regular practice of preventing mosquito presence and mosquito bites in order to prevent dengue fever.\n\nKnowledge of dengue was measured by asking questions related to disease vector mosquitoes, mode of disease transmission, and breeding places of mosquitoes. Questions related to knowledge were asked before the questions of preventive measures to avoid bias. Different mosquito prevention measures were used to investigate their practices, which included checking the house and surroundings for removing stagnant water, use of mosquito nets at night and even during daytime sleep, and mosquito killing/avoiding devices.\n\nThe preliminary draft of the questionnaire was tested before finalizing. A few inappropriate questions were omitted, resulting in a total of 20 questions in the final questionnaire (Extended data, Rahman, 2022b). The questionnaire was designed in the English language and translated into Bengali (mother tongue of the participants) during interview. In case of children, data were collected from parents or a guardian.\n\nWe developed a numeric scoring system to assess general knowledge and awareness of dengue vectors. Knowledge-related questions were scored as 1 point for the correct or “Yes” answer, and 0 for the wrong or “No” answer. The maximum total score was 8 and participants were ranked into two groups: good knowledge (score 5 to 8) and poor knowledge (score 1 to 4). However, questions about prevention practices were scored mostly along a four-point scale ranging from 0 to 3, as regularly (score 3), frequently (score 2), occasionally (score 1), and never (score 0). Thus, the maximum total score was 20 and divided the participants under strong practice (11 to 20 points) and weak practice (1-10 points). The respondents’ information was stored on the computer with a password and was accessible only to the investigators. People who failed to respond to all questions or who left before completing the interview were excluded.\n\nFirst, the demographic variables were compared between the two groups of participants. Then, the associations between the ranks and socio-demographic characteristics were investigated using Chi-Square and Fisher exact probability tests. Non-overlapping 95% CI and a p-value ≤5% were considered as statistically significant. Finally, logistic regression was used to identify determinants influencing the knowledge and prevention practice of dengue among the people.\n\nThis study was conducted by following the Declaration of Helsinki, and the project protocol was approved by the Ethics Committee of the Chattogram Veterinary and Animal Sciences University, Bangladesh (permit reference number: CVASU/Dir(R&E) EC/2020/169/10, Date: 21/07/2020.\n\n\nResults\n\nIn this study, a total of 300 respondents were surveyed, of which 150 were dengue patients and the remaining 150 were the general people without experience of being infected with dengue. Table 1 compares the profiles of the two group of participants. All demographic variables showed a significant difference between the groups. A higher number of cases was found in young people aged below 15 years (77%) followed by 16 to 30 years (68%). On the other hand, middle-aged to older people were mostly found in the non-case group: 60% between 31 and 45 years, 73% between 46 and 60 years, and the highest, 86.6% were more than 60 years old. Male numbers were higher in cases (57.4%) and females were mostly non-case (61.5%). Among different occupation categories were government officials (74.3%), and students (73.4%) were frequently recorded in the case group. Housewives (70%) and unemployed people (69.6%) were mostly non-case. Cases were mostly from urban (65%) and semi-urban (66.2%) areas while 88.4% non-cases were from rural areas. People who lived or worked with dengue patients were mostly in the case group (89.1%). Among all participants, permanent residents showed a higher proportion of non-cases (54.3%) while most of the temporary residents (73.9%) were found as cases.\n\nTable 2 compares the level of knowledge and practice of prevention among participants. Good knowledge was found higher (72%) among the dengue-infected than the non-infected group (51.3%). The difference of knowledge level between the groups was significant (p< 0.05). On the other hand, participants of both groups were mostly demonstrated to have weak (87.3% cases and 91.3% non-cases) practices for dengue vector prevention.\n\nOccupation and geographic location of the people showed a significant effect on their level of knowledge (see Table 3). The people living in urban areas (71.4%) were more knowledgeable than the semi-urban and rural area (p-value=0.00). Among the different occupations, students (78.1%), government officials (77.1%), and businessmen (66.7%) possessed comparatively better knowledge than others.\n\nTable 4 shows that urban people had a 2.20 times higher knowledge compare to semi urban. The level of knowledge was 3.39 times stronger among students and 3.17 times stronger within government officials in comparison with workers. Moreover, the binary analysis shows a significant association of permanent residents who had 2.01 times with a strong knowledge compared to the people who lived in temporary homes.\n\nOur study found a strong association between the geographical location and the practice level where 25.7% of the people living in the semi-urban area were reported to have higher preventive practices than the people who lived in the rural or urban area. Additionally, living with dengue patients was also significantly associated with good preventive practices with a p-value of 0.00 (see Table 5).\n\nIn Table 6, showing the binary regression results, it was found that the people who lived in semi-urban were practicing 3.19 times (CI=0.97-10.52) stronger preventive measures compared to those living in rural areas. Furthermore, respondents who had never lived with dengue patients were significantly less practiced for different mosquito preventive measures than those who were experienced with living with dengue patients. In addition to that, males were reported to practice almost 1.02 times more preventive measures than females.\n\nThe responses of participants for different knowledge-related questions are presented in Table 7 and show significant differences among responses between the two groups. A total of 50.3% of participants with good knowledge correctly answered that the dengue fever caused by a virus while 92.2% of those with poor knowledge had no idea regarding the causal agent. A total of 99.5% participants with good knowledge recognized that dengue was transmitted by mosquitoes, and 82.2% of them knew the name Aedes spp. and that this mosquito breeds in clean water. Most of the participants (94.6%) having good knowledge mentioned that dengue fever is treatable whereas 21.1% of participants possessing poor knowledge did not know the availability of treatment for dengue.\n\nTable 8 presents significant differences in the responses of prevention practices-related questions between strong and weak-practice groups of participants. Most of the participants in the strong practice group regularly used mosquito nets at night (84.4%) and in daytime as well (12.5% regularly and 31.3% frequently). A higher number of people belonging to the strong-practice group frequently (56.3%) and regularly (40.6%) used different mosquito killing devices in their home, while on the other hand, 22% people from the weak practices group never used any mosquito killing devices. A total of 29% of participants having strong practices checked and cleaned stagnant water daily in and around their house, whereas 50.4% of participants with weak practices never checked stagnant water.\n\nFigure 1 shows that people mostly (58%) used coil to prevent mosquitoes. However, 15.33% used both coil and mosquito killing spray. A small number of people (1.33%) also used electric appliances in their houses.\n\n\nDiscussion\n\nIn this study we have investigated the level of basic knowledge on dengue vectors and practice of preventive measures in dengue-infected and non-infected people. Both groups were significantly different in terms of socio-demographic characteristics such as age, gender, occupation, geographic location, living with dengue patients, and residence types. Among different occupational categories, state workers and students were in the case group. Housewives and unemployed people were mostly found in the non-case group.\n\nIn light of the findings of this study, the level of knowledge was high among the participants of both groups. This finding was consistent with a similar study conducted over the same period throughout Bangladesh, which found more than half of the participants (65.9%) had sufficient knowledge regarding dengue (Hossain et al., 2021). Another study conducted in Philippines also demonstrated that 61.45% of the people had a good knowledge on causes, signs and symptoms, method of transmission, and prevention strategies of dengue (Yboa and Labrague, 2013). However, a cross-sectional survey in Indonesia showed opposite findings, where more than half of the participants had no prior knowledge of dengue fever (Harapan et al., 2018). Despite the fact that the majority of participants of both groups had a good understanding on the dengue vector, they were found to have weak practices in terms of preventive measures. Previous literature supports these findings by stating that over 75.7% of cases and 73.6% of controls were totally unaware of how to prevent dengue (Degife et al., 2019). This indicates that having a good knowledge does not always ensure a good practice. Nevertheless, a study in Vietnam showed a completely opposite result, revealing people with good knowledge of dengue fever were found to have good prevention practices and awareness with a co-efficient = 0.44; 95% CI = 0.35–0.53 (Nguyen et al., 2019)\n\nAmong participants, the young (16-30 years) had a good knowledge of dengue vectors in comparison to older people, which was supported by a previous study (Patil et al., 2020). The similar result found in another study done in Cambodia in 2018, where younger people aged less than 35 years showed strong knowledge about dengue vector, breeding place, and prevention (Kumaran et al., 2018). In contrast, the literature also indicated that people over 40 years have a better understanding of dengue fever than younger people (Roslan et al., 2020). The poor knowledge among middle-aged to older people (31 to 60 years) found in this study could be explained by the fact that they may be less concerned about disease.\n\nResults showed relatively good knowledge of dengue infection among males. The results of our binary logistic regression show that males had 1.49 times higher knowledge than females, however, the effect was statistically non-significant. This finding was concordant with a previous study conducted in Malaysia (Zamri et al., 2020). Likewise, a study in Ethiopia found an association between gender and the knowledge level of participants (Yusuf and Ibrahim, 2019). On the other hand, a study in Yangon found gender was a significant factor and females had more knowledge about dengue (p-value <0.01) (World Health Organization, 2004).\n\nWe found a significant association between occupation, and particularly students (OR=3.39) and government workers (OR=3.17), with the knowledge score. A prospective study in Northern Thailand also revealed a significant association with profession, where students had a 10.6 times higher knowledge on dengue compared to housewives or unemployed people (Van Benthem et al., 2002). Another study in Ethiopia also stated that the type of profession was associated with the knowledge of dengue and found higher levels of knowledge among public health officers (AOR= 38.79) and physicians (AOR=6.15) than nurses (Yusuf and Ibrahim, 2019). However, a study in Vietnam found no association of occupation with the knowledge level (Nguyen et al., 2019).\n\nGeographical location is another significant factor that was found to be strongly associated with the knowledge level of participants. The binary logistic regression analysis showed that urban people had 2.20 times more knowledge (CI= 1.15-4.20) compared to those who lived in semi-urban area. Similarly, a previous study also demonstrated that the city residents were 1.09 times more likely to have good knowledge than the people living in a village or semi-urban (CI=0.635–1.897) (Hossain et al., 2021). Another cross-sectional study also investigated that urban residents has better knowledge about the Aedes mosquito, breeding site, symptoms of dengue fever than the rural residents (Singru et al., 2013). The literature also revealed associations with topography: people living in high land have five times less knowledge than those who lived in low land in Kathmandu, Nepal (Dhimal et al., 2014). Besides, a binary regression showed that permanent residents of the city had significantly better knowledge (OR=2.01) than the temporary residents.\n\nAlthough most of the participants were found to have a better understanding of dengue vectors, their engagement in terms of practicing preventive measures was very inadequate. A cross-sectional study in Indonesia found a significant effect of age (above 41 years) and gender (female) on better prevention practices than younger and males (Rakhmani et al., 2018). A Malaysian study also discovered that participants between the ages of 25 and 40 had a higher preventative behavior score than those between the ages of 18 and 24 (Roslan et al., 2020). However, we did not find any significant effect of age and gender on the level of prevention practice of respondents which was found to be consistent with a study conducted in Malaysia (Zamri et al., 2020). However, binary logistic demonstrated that males were 1.02 times more likely to have less awareness than females.\n\nFurthermore, logistic analyses revealed that housewives practiced preventive measures 2.09 times more than workers; however the association was not significant. The result was consistent with a paper from India in which homemakers were reported to have greater awareness regarding dengue prevention than other professionals (Chellaiyan et al., 2017). Studies conducted in Vietnam and Ethiopia found a significant association between occupation and the awareness of dengue prevention (Yusuf and Ibrahim, 2019; Nguyen et al., 2019).\n\nWe found a significant effect of location of participants on prevention practices, and people living in semi-urban area practiced 3.19 times more mosquito prevention methods than people in rural area. On the other hand, a multinominal regression analysis reported that people living in suburban areas were 1.01 times less aware of preventive measures than the people living in city (CI=0.67–1.53) (Harapan et al., 2018).\n\nMore than half of our respondents positively replied that dengue is transmitted by mosquito and could also mention the name of vector mosquito. Similar literature also reported that nearly 89% of participants identified that Aedes mosquitoes transmit dengue in human (Chellaiyan et al., 2017). However, a study in Nepal observed opposite results, where only 19% of the participants properly named Aedes mosquito (Dhimal et al., 2014).\n\nFurthermore, most of the participants marked that Aedes mosquitoes bite in daytime which was supported by a previous study that also found 43.1% Bangladeshi participants knew that Aedes mosquitoes bite during the dawn or dusk (Hossain et al., 2021). Similarly, a study in Thailand also revealed that most of the people were aware about the biting time of the dengue-causing mosquito (Dégallier et al., 2000; Van Benthem et al., 2002). On the contrary, previous studies in Laos and Nepal also showed that these majority of the research participants did not have proper knowledge about the biting time of Aedes mosquito (Mayxay et al., 2013; Dhimal et al., 2014).\n\nFurthermore, results showed that participants were aware that personal contact with dengue patients did not transmit dengue, which contradicted the findings of a previous study that found 56% of people had no enough knowledge about this (Dhimal et al., 2014). Regarding the breeding site, half of the research population stated that Aedes mosquitoes can grow in clean water, which was similar to a study in Laos, while studies also reported that 71% of people believed containers with filthy water can be a breeding site for dengue vectors (Malhotra et al., 2014) and had limited knowledge about the breeding site (Syed et al., 2010). A study conducted in India found that 40% of the respondents had proper knowledge regarding the breeding site of the vectors (Chellaiyan et al., 2017). We found that, most of the participants with good knowledge answered correctly that dengue fever has a treatment, which was concordant with the finding of a previous study in Bangladesh that stated that 89.6% of people were aware about the treatment of dengue (Hossain et al., 2021).\n\nAlong with dengue patients, we interviewed an equal number of non-infected people which was a great strength of this study. Furthermore, data were collected from cases within two weeks after patient release from hospital which reduced the chance of recall bias. The limitation of the study was the fewer number of questions used for scoring of knowledge and prevention practice. Besides, the data was collected through telephone interview which caused a higher number of incomplete responses.\n\n\nConclusions\n\nAssessing the existing perception of people is crucial for confirming active community engagement in dengue vector control program. We found good knowledge among most of the participants on dengue vector, however, the practice of mosquito vector prevention was weak. Urban people had good knowledge on dengue vectors while the people living in semi-urban areas were found to strongly practice mosquito prevention methods. Among different occupation groups, students and government officials possessed good knowledge. The results indicated that having a good knowledge does not always ensure a good practice in humans. This study could help policy makers and regulatory bodies for controlling dengue through promoting and monitoring good vector prevention practice among the people.\n\n\nData availability\n\nFigshare: Knowledge and prevention practice regarding dengue fever, https://doi.org/10.6084/m9.figshare.18093836 (Rahman, 2022a).\n\nThis project contains the following underlying data:\n\n- dengue knowledge-raw data.xlsx (individual survey answers and scores)\n\nFigshare: Questionnaire on Knowledge and Prevention Practice of Dengue Vector in Chattogram, Bangladesh, https://doi.org/10.6084/m9.figshare.19064078.v1 (Rahman, 2022b).\n\nThe project contains the following extended data:\n\n- Questionnaire.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgment\n\nThe authors acknowledge the support of the department of medicine of Chittagong medical college hospital and the Bangladesh institute of tropical and infectious diseases for providing the patient’s record data.\n\n\nReferences\n\nAbbasi A, Abbas K, Arooj S, et al.: Dengue Fever: A Statistical Analysis Regarding Awareness about Dengue among University Students in Azad Kashmir. 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Public Health. 2019; 16: 976. PubMed Abstract \| Publisher Full Text\n\nPatil PJ, Patil VS, Chaudhari RD, et al.: Awareness, Knowledge and Attitude about Dengue Among Society. Int. J. Curr. Pharm. Res. 2020; 36–40. Publisher Full Text\n\nRafi A, Mousumi AN, Ahmed R, et al.: Dengue epidemic in a non-endemic zone of Bangladesh: Clinical and laboratory profiles of patients. PLoS Negl. Trop. Dis. 2020; 14: e0008567–e0008514. PubMed Abstract \| Publisher Full Text\n\nRahman MS, Mehejabin F, Rashid R: A community based case-control study to determine the risk factors of dengue fever in Bangladesh. medRxiv. 2021a. Publisher Full Text\n\nRahman MS, Faruk MO, Tanjila S, et al.: Entomological survey for identification of Aedes larval breeding sites and their distribution in Chattogram, Bangladesh. Beni-Suef Univ. J. Basic Appl. Sci. 2021b; 10: 1–11. Publisher Full Text\n\nRahman MS, Mehejabin F, Rashid R: Knowledge and prevention practice regarding dengue fever-raw data with score.xlsx. figshare. Dataset. 2022a. Publisher Full Text\n\nRahman MS, Mehejabin F, Rashid R: Questionnaire on Knowledge and Prevention Practice of Dengue Vector in Chattogram, Bangladesh. figshare.2022b. Online resource. Publisher Full Text\n\nRakhmani AN, Limpanont Y, Kaewkungwal J, et al.: Factors associated with dengue prevention behaviour in Lowokwaru, Malang, Indonesia: A cross-sectional study. BMC Public Health. 2018; 18: 1–6. Publisher Full Text\n\nRoslan MA, Ngui R, Vythilingam I, et al.: Survey of Dengue Knowledge and Prevention Practices Associated with Sociodemographic Status: A Cross-Sectional Study Among the Community Living in an Urban Area of Selangor, Malaysia. J. Am. Mosq. Control Assoc. 2020; 36: 115–119. PubMed Abstract \| Publisher Full Text\n\nSingru S, Bhosale S, Debnath D, et al.: Study of knowledge, attitude and practices regarding dengue in the urban and rural field practice area of a tertiary care teaching hospital in Pune, India. undefined. 2013; 6: 374. Publisher Full Text\n\nSyed M, Saleem T, Syeda UR, et al.: Knowledge, attitudes and practices regarding dengue fever among adults of high and low socioeconomic groups. J. Pak. Med. Assoc. 2010; 60: 243–247. PubMed Abstract\n\nVan Benthem BHB, Khantikul N, Panart K, et al.: Knowledge and use of prevention measures related to dengue in northern Thailand. Tropical Med. Int. Health. 2002; 7: 993–1000. PubMed Abstract \| Publisher Full Text\n\nWHO: Dengue and severe dengue.2020. Accessed 18 Feb 2021. Reference Source\n\nWilder-Smith A, Ooi EE, Horstick O, et al.: Dengue. Lancet. 2019; 393: 350–363. Publisher Full Text\n\nWorld Health Organization: Community-based Assessment of Dengue-related Knowledge among Caregivers.2004. Accessed 18 Nov 2021.Reference Source\n\nWorld mosquito program: Dengue\|World Mosquito Program.2021. Accessed 16 Oct 2021.Reference Source\n\nYboa BC, Labrague LJ: Dengue Knowledge and Preventive Practices among Rural Residents in Samar Province, Philippines. Am. J. Public Heal. Res. 2013; 1: 47–52. Publisher Full Text\n\nYusuf AM, Ibrahim NA: Knowledge, attitude and practice towards dengue fever prevention and associated factors among public health sector health-care professionals: in Dire Dawa, eastern Ethiopia. Risk Manag. Healthc. Policy. 2019; 12: 91–104. PubMed Abstract \| Publisher Full Text\n\nZamri SNZBM, Rahman NAA, Haque M: Knowledge, Attitude, and Practice Regarding Dengue among Students in a Public University in Malaysia. Bangladesh J. Med. Sci. 2020; 19: 245–253. Publisher Full Text" }	[ { "id": "184377", "date": "17 Aug 2023", "name": "Prasad Liyanage", "expertise": [ "Reviewer Expertise Epidemiology and Public Health with special orientation towards vector borne 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\nIs the work clearly and accurately presented and does it cite the current literature?\nThe article attempts to address an important knowledge gap in the literature for implementing effective and sustainable vector control intervention. Dengue vector control demands high degree of community acceptability and participation for its success. Understanding the perception of the target community about the disease, its prevention and control is an essential but usually neglected component of the intervention process.\nI would like to draw the attention of authors on following points for the purpose of improving the clarity of the information.\nIntroduction section:\nThe description of the disease transmission patterns, and different outcomes is too general. I would suggest the authors to provide more specific and quantifiable information on the blood born and vertical transmission rates as these events are extremely rare. Please specify how often the dengue fever can present with dengue shock syndrome and dengue hemorrhagic fever that is relevant to your context.\nEpidemiologically, it is always intuitive to provide incidence along with the number of cases. The readers can compare the disease burden in your setting with global or their local situation. Please revise the second paragraph providing the incidence rates. Please also be consistence with the cases and incidence.\nPlease emphasize the role of population immunity and introduction or re-introduction of different DENV serotypes which are considered to be major drivers for the dengue transmission.\nIs the study design appropriate and does the work have academic merit?\nPlease rationalize why you select Chattogram district for your study.\nThere are two fundamental methodological issues in the study design identified which can hinders the scientific validity of the results.\nRepresentativeness to the population under consideration. The authors mention that “Contact information of non-case participants was collected from the people who visited the outpatient department of the same hospitals”. It is questionable whether the sample selected as non-dengue (which is the comparator for all other variables analyzed) is representative of the Chattogram district community. Some people were not ill to attend to the hospital during the study period and some may not have access.\n\nNon-matching issue of the comparator. When comparing the knowledge, attitudes and practices, it is fundamental to use age and sex-matched comparator groups to avoid selection bias. As demonstrated by the twisted ratios of participants at the extreme age groups (Table 1; Age <=15 years and age >=65 years), it is clear that the proportions between dengue and non-dengue were not only affected by the transmission pattern of dengue during the study period but the generally increasing tendency of presenting to the outpatient departments with increasing age. The comparator group should at least have an equal number of cases for each age group and sex.\nAre sufficient details of methods and analysis provided to allow replication by others?\nAuthors should pay attention to providing sufficient details on the measures taken to minimize the confounding bias and information bias on the selected sample. This is particularly valid because of my above comments on the study design. Reproducibility and generalizability are of major concern in the present context of the study design.\nIf applicable, is the statistical analysis and its interpretation appropriate?\nWhile the authors have provided some insights into the knowledge and preventive practices related to dengue among the people living in the Chattogram district, comparing the dengue and non-dengue groups requires careful consideration of potential biases. I strongly recommend the authors pay attention to the above methodological issues and provide a detailed account of how they addressed them in their next revision.\nAre all the source data underlying the results available to ensure full reproducibility?\nPlease refer to my response above.\nAre the conclusions drawn adequately supported by the results?\nPlease refer to my response above. Major revisions are needed before publishing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "212053", "date": "25 Oct 2023", "name": "Thang Nguyen-Tien", "expertise": [ "Reviewer Expertise Public Health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSome questions/comments to consider:\nOverall, this manuscript needs the proofing in English\nUse \"preventive practice\" for the whole manuscript\nIntroduction section\nAccording to a study conducted in Malaysia, a lack of awareness about dengue transmission and prevention strategies can enhance the risk of infection (Nguyen et al., 2019). --> Not from Malaysia, it should be Vietnam\n\nPrevious knowledge, attitude, and practice (KAP) studies mainly focused on clinical aspects of dengue fever while the vector perspectives remained neglected. --> Add the reference\nMethods section\nA comprehensive review of the literature was conducted to identify the factors determining the level of knowledge and dengue prevention practices among the people. --> What is the implication to put this sentence in the study design part? Suggest to remove\n\nExplain why choosing Chattogram district for this study? Better to describe about some general characteristics of this district\n\nHow was the pretest for questionnaire (with whom, how many...)?\n\nScoring system: Why using the cut-off points of 50% total score? I think it's better to use other scoring system that some previous studies indicated or just use K&P score as the continuous variables\n\nResearch ethic: The ethical approval was obtained in 2020 but data collection period was conducted from 2019?\nResults section\nAny possible confounders? Please indicate in the data analysis part\n\nCan put knowledge score variable into the logistic regression model of preventive practice score to see whether it's correlated or not\nDiscussion section\nAdd more implications for each discussed 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? Yes\n\nAre 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": "172838", "date": "09 Sep 2024", "name": "Sulistyawati Sulistyawati", "expertise": [ "Reviewer Expertise Public Health", "epidemiology and health system research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research is good, but data collection was done several years ago, so it is necessary to add information about the currently existing.\n1. Methodology: This study employs a phone survey susceptible to respondent bias. Please explain the procedures for recruiting respondents, as well as how researchers ensure that the respondents they seek are those who receive phone calls. 2. The researcher stated that the sample was collected from two tertiary care hospitals; how can the researcher ensure that the response to that area is representative? There is no explanation for the location settings used in the background, and the rationalization of the research area is not well-defined. 3. The method stated that the study was conducted between July and December 2019. The researcher stated that the cases were recent cases; what is the definition of recent cases? It is best to record the case's date. 4. The method by which each item of the questionnaire is scored Please include the questionnaire as an appendix.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-146
https://f1000research.com/articles/10-1070/v1	22 Oct 21	{ "type": "Brief Report", "title": "Prospective cohort study of the predictive value of inflammatory biomarkers over clinical variables in children and young people with cancer presenting with fever and neutropenia", "authors": [ "Bob Phillips" ], "abstract": "Introduction Fever during chemotherapy induced neutropenia is a common and potentially life-threatening complication of the treatment of childhood cancer. Predictions of poor outcome could be enhanced by incorporating serum biomarkers of inflammation at presentation and reassessment. Methods A prospective cohort study was conducted of children under 18 years old, being treated for cancer or a cancer-like condition, who presented with fever (≥ 38.0°C) and neutropenia (neutrophil count < 0.5*109/L). Clinical features were recorded, along with three experimental inflammatory biomarkers: procalcitonin (PCT), interleukin-6 (IL-6) and interleukin-8 (IL-8). Outcomes included serious medical complications (SMC): any infection related mortality, critical care and organ support, severe sepsis, septic shock, significant microbiologically defined infection, or radiologically confirmed pneumonia. Results Biomarker assessments were undertaken in 43 episodes of fever and neutropenia, from 31 patients aged between four months and 17 years old (median six years): 20 were female and 22 had acute leukaemia. Five episodes of SMC were noted. PCT, IL-6 and IL-8 had poor individual discriminatory ability (C-statistic 0.48 to 0.60) and did not add to the value of clinical risk stratification tools. Insufficient data were collected to formally assess the value of repeated assessments. Conclusions Incorporating serum biomarkers of inflammation at presentation of episodes of fever with neutropenia in childhood does not clearly improve risk stratification. Repeated assessments over time may be of value.", "keywords": [ "febrile neutropenia", "neutropenic sepsis", "childhood cancer", "inflammatory biomarkers", "IL6", "IL8", "procalcitonin" ], "content": "Introduction\n\nInfection, frequently presenting as fever during chemotherapy induced neutropenia, is a common and potentially life-threatening complication of the treatment of childhood cancer. Modern management approaches have promoted the use of clinical decision rules to stratify patients at low risk of serious medical complications during febrile neutropenia (FN), enabling them to be safely managed with less intensive therapy as an outpatient.1\n\nIn addition to the role of clinical risk stratification, there is an increased desire to use modern biochemical markers of inflammation in predicting the risk of severe sepsis. There is increasing interest in inflammatory biomarkers such as procalcitonin (PCT), an inflammatory marker that has been shown to rise in response to bacterial infection in non-immunosuppressed children,2 and various cytokines including interleukin (IL)-6 and IL-8 to improve the diagnostic accuracy of a prediction rule in children with FN.3\n\nA systematic review and meta-analysis of the discriminatory ability of biomarkers in children with FN4,5 concluded that while several small studies suggest PCT, IL-6 and IL-8 may be valuable for predicting severe infection in children with FN, the true impact remains unknown. A smaller number of studies explored the role of serial (i.e., 0 h, 12–24 h, 48 h) biomarkers to detect documented infection or sepsis. In one study, the difference between mean C-reactive protein (CRP), PCT and IL-8 at 24 hours in children with and without sepsis was more pronounced than at presentation.6 These data suggest the optimal value for prediction may be made by incorporating biomarkers at early reassessment (i.e., within 12–24 h or 24–48 h), rather than at presentation.\n\nThis study aimed to undertake a focused analysis of three promising inflammatory biomarkers: PCT, IL-6 and IL-8 in both initial and value of serial testing and their additional discriminatory value above routine clinical features using the PICNICC model7 and AUS-score.8\n\n\nMethods\n\nThis study took place in Leeds Children’s Hospital between March 2016 and March 2018. It recruited patients on presentation of FN or pre-enrolled them during routine appointments where they, or their parents or guardians, affirmed their consent in written form. In addition to formal information sheets, a link to an animated video summary of the research was provided https://www.youtube.com/watch?v=Z1AXzJqatds.9 Ethical approval was given by the Leeds West NHS Research Ethics Committee [15/YH/0357].\n\nChildren could be included who were: younger than 18 years old, who had cancer, or received a stem cell transplant, or who had Langerhans cell histiocytosis in need of cytotoxic chemotherapy, or haemophagocytic lymphohistiocytosis undergoing active treatment, or severe aplastic anaemia, and attended with febrile neutropenia. Fever was defined as temperature ≥ 38.0°C and neutropenia, an absolute neutrophil count ≤ 0.5 g/L.\n\nAll patients were admitted to hospital and managed according to institutional FN pathways; these consisted of full evaluation and empiric piperacillin/tazobactam (or suitable alternatives) until afebrile 48 hours and with no other reason for antimicrobials, regardless of neutrophil count. Inflammatory biomarkers, with the exception of CRP, were analysed in batches and their results masked from the treating team. Using this unselective approach, with treatment unaffected by the results of the biomarker analyses, we minimized the biases which can arise through cherry-picking of patients and altering medical treatment on the basis of the test under evaluation (selection and incorporation biases).\n\nDemographic data, variables and outcomes included core items as devised by the International Paediatric Fever in Neutropenia Working Group (see Extended Data, Table 1)9. Clinical assessments and blood samples for biomarkers were taken at presentation (within 12 h of fever or admission) and daily until the end of the FN episode or discharge as part of usual clinical care.\n\nThe primary outcome measure was ‘serious medical complication (SMC)’10 (defined by any of (i) infection related mortality (ii) admission to ICU/HDU/other ward/unit for organ support (iii) severe sepsis or (iv) septic shock) or significant microbiologically defined infection, or radiologically confirmed pneumonia. Secondary outcomes measures were the component parts of the primary outcome, death within 30 days, infection related mortality, clinically defined infection, infection with antibiotic-resistant bacteria and relapse of primary infection. Initial power calculations estimated ~400 episodes would detect an additional benefit C-statistic discriminatory of +0.10.\n\nResults were analysed descriptively, and assessment made of the individual discriminatory value of the inflammatory biomarkers using the C-statistic for SMC, and their additional value to clinical prediction rules (PICNICC prediction and AUS-score). Analyses were undertaken using base R version 3.6.0 (R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria) (RRID:SCR_001905) and the package pROC (pROC: an open-source package for R and S+ to analyze and compare ROC curves) Further analysis of the results was planned to be by hierarchical logistic regression modelling of episodes within patients, assessing the predictive value of clinical and then biomarkers of inflammation at admission, day one and subsequent timepoints. Additional assessments of the sensitivity and specificity of the markers, alone and in combination, were also proposed.\n\n\nResults\n\nData was collected from 65 patients with an episode of fever and neutropenia. Of those, 43 episodes in 31 patients had biomarker samples taken, with 31 episodes with ‘day one’ data. The distribution of diagnoses is shown in Table 1. The patients ranged from four months to 17 years old (median six years), and 20 were female.\n\nOf these 31 episodes, five were assigned an SMC: two significant viral infections with oxygen requirement, and one each of: Fusobacterium blood stream infection, Escherichia coli urinary tract infection, and culture-negative severe sepsis requiring fluid boluses. No patient received intensive care support or died. There were eight non-serious viral infections and one non-serious central line colonization.\n\nAll biomarkers performed poorly in distinguishing those who developed an SMC (see Figure 1), with C-statistic estimates ranging from 0.48 to 0.60 (see Table 2). The biomarkers were ineffective in improving the discriminatory value of the PICNICC risk prediction (change in C-statistic: −0.10 to +0.04), and only marginally useful in improving the simpler AUS-score (+0.04 to +0.18) (See Extended Data, Table 2)9 Given the paucity of data and lack of diagnostic value, no complex analyses were undertaken.\n\nCRP = C-reactive protein; PCT = procalcitonin; IL-6 = interleukin-6; IL-8 = interleukin-8.\n\nCRP = C-reactive protein; PCT = procalcitonin; IL-6 = interleukin-6; IL-8 = interleukin-8.\n\nThe biomarker levels concurred better with the physician assessment of “severe” clinical illness, though in no case was this statistically significantly different (see Table 3, p > 0.10).\n\nCRP = C-reactive protein; PCT = procalcitonin; IL-6 = interleukin-6; IL-8 = interleukin-8.\n\nRepeated measures of the biomarkers were available in 25 episodes. Development of SMC did not occur in patients who presented with non-severe symptoms and consistently low inflammatory biomarkers despite ongoing fevers. Of those with SMC, 4/5 had reductions in biomarker levels as their infection resolved; they stayed high in the one case of culture negative severe sepsis.\n\n\nDiscussion\n\nThis prospective study of inflammatory biomarkers in paediatric febrile neutropenia found little support for their use as indicators of covert infection. Technical and administrative challenges limited the number of samples collected during the study, despite enthusiasm from the patients and their families. This adds to the body of evidence describing a relatively limited role in initial stratification.\n\nSerial use of these markers, where they can be used to suggest an infection is under control, or controlled, is an area of active testing. PCT has been studied in several patient groups to diagnose sepsis and monitor response to treatment. A systematic review11 and subsequent large (>1500 patients) RCT with pragmatic study design examining PCT-guided decision-making in neonates (NeoPIns) with suspected infection and critically ill adults on ICU (SAPS) found significant reductions in antibiotic duration in the PCT arm.12,13 Two large UK studies looking at the same question in adults and children with suspected or proven serious bacterial infections are ongoing.14,15 These studies specifically exclude immunocompromised patients such as children with cancer, and work exploring this is required to enhance patient experience and reduce antimicrobial resistance.16–18\n\nThe results of this study should dissuade clinicians from routinely using inflammatory biomarkers in making initial stratification of children with febrile neutropenia. The onward investigation of their serial use in antimicrobial stewardship should be pursued within carefully monitored studies.\n\n\nData availability\n\nThe data for this study contains the following elements, which when combined would make the patients identifiable given the rarity of childhood cancer in the identified geographical area and time of this study:\n\nDemographic data: (ii) episode date; (iii) age; (iv) cancer diagnosis & treatment.\n\nFN episode data: (i) inpatient or outpatient onset; (ii) time of start of temperature; (iii) time of presentation.\n\nA reduced dataset taking out all items to produce sufficient anonymity (for sensitive data; childhood cancer) would severely limit their utility for researchers.\n\nThe patients and families who took part in this study, and the Research Ethics Committee who granted permission for it, agreed the data should be available for sharing in ethically approved secondary use projects. Such studies are typically individual participant data meta-analysis collaboratives. Anyone who has such an approved project, investigating aspects of paediatric febrile neutropenia and biomarker profiles, is encouraged to approach the author at bob.phillips@york.ac.uk for access to the dataset.\n\nOpen Science Framework (OSF): Extended data for ‘Prospective cohort study of the predictive value of inflammatory biomarkers over clinical variables in children and young people with cancer presenting with fever and neutropenia’, https://www.doi.org/10.17605/OSF.IO/CVFZB.9\n\nThis project contains the following extended data:\n\n• Extended Data Tables.docx (Extended Table 1: Data items collected and Extended Table 2: AUC-ROC (C-statistic) values for biomarkers to distinguish patients with SMC)\n\n• Consent Video (mp4 format)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nWritten informed consent for publication of the patients’ details was obtained from the patients/parents/guardian of the patient.", "appendix": "References\n\nKlastersky J, Paesmans M, Rubenstein EB, et al.: The Multinational Association for Supportive Care in Cancer risk index. J. Clin. Oncol. 2000; 18(16): 3038–51. PubMed Abstract \| Publisher Full Text\n\nIrwin AD, Carrol ED: Procalcitonin. Arch. Dis. Child. Educ. Pract. Ed. 2011; 96(6): 228–33. PubMed Abstract \| Publisher Full Text\n\nHaeusler GM, Slavin MA: Complications of sepsis: the role of risk prediction rules, biomarkers and host genetics. Expert Rev. Anti. Ther. 2012; 10(7): 733–5. PubMed Abstract \| Publisher Full Text\n\nPhillips RS, Wade R, Lehrnbecher T, et al.: Systematic review and meta-analysis of the value of initial biomarkers in predicting adverse outcome in febrile neutropenic episodes in children and young people with cancer. BMC Med. 2012; 10: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaeusler GM, Carlesse F, Phillips RS: An Updated Systematic Review and Meta-Analysis of the Predictive Value of Serum Biomarkers in the Assessment of Fever during Neutropenia in Children with Cancer. Pediatr. Infect. Dis. J. 2013; 32(10): e390–6. [published Online First: 2013/05/16].PubMed Abstract \| Publisher Full Text\n\nSantolaya ME, Alvarez AM, Aviles CL, et al.: Predictors of severe sepsis not clinically apparent during the first twenty-four hours of hospitalization in children with cancer, neutropenia, and fever: a prospective, multicenter trial. Pediatr. Infect. Dis. J. 2008; 27(6): 538–43. PubMed Abstract \| Publisher Full Text\n\nPhillips RS, Sung L, Ammann RA, et al.: Predicting microbiologically defined infection in febrile neutropenic episodes in children: global individual participant data multivariable meta-analysis. Br. J. Cancer. 2016; 114(12): e17. [published Online First: 2016/05/27].PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhillips B, Morgan JE: Meta-analytic validation of new 'AUS' febrile neutropenia risk score. Pediatr. Blood Cancer. 2021; 68(1): e28580. [published Online First: 2020/07/28].PubMed Abstract \| Publisher Full Text\n\nPhillips B: NIHR-PDF Biomarkers. OSF. 2021. Publisher Full Text\n\nGoldstein B, Giroir B, Randolph A, et al.: International pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics. Pediatr. Crit. Care Med. 2005; 6(1): 2–8. PubMed Abstract \| Publisher Full Text\n\nWestwood M, Ramaekers B, Whiting P, et al.: Procalcitonin testing to guide antibiotic therapy for the treatment of sepsis in intensive care settings and for suspected bacterial infection in emergency department settings: a systematic review and cost-effectiveness analysis. Health Technol. Assess. (Winch. Eng.). 2015; 19(96): 1–236. [published Online First: 2015/11/17].PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStocker M, van Herk W , El Helou S, et al.: Procalcitonin-guided decision making for duration of antibiotic therapy in neonates with suspected early-onset sepsis: a multicentre, randomised controlled trial (NeoPIns). Lancet (London, Eng.). 2017; 390(10097): 871–81. [published Online First: 2017/07/18].PubMed Abstract \| Publisher Full Text\n\nde Jong E , van Oers JA , Beishuizen A, et al.: Efficacy and safety of procalcitonin guidance in reducing the duration of antibiotic treatment in critically ill patients: a randomised, controlled, open-label trial. Lancet Infect. Dis. 2016; 16(7): 819–27. [published Online First: 2016/03/08].PubMed Abstract \| Publisher Full Text\n\nADAPT-Sepsis: Biomarker-guided duration of antibiotic treatment for sepsis: ISRCTN47473244.\n\nBiomarker-guided duration of Antibiotic Treatment in Children Hospitalised with confirmed or suspected bacterial infection (The BATCH Trial): ISRCTN 11369832.\n\nPublic Health England: Start Smart - Then Focus: Antimicrobial Stewardship Toolkit for English Hospitals. PHE Publications Gateway 2014828. March 2015.\n\nNational Institute for Health and Care Excellence: Procalcitonin testing for diagnosing and monitoring sepsis (ADVIA Centaur BRAHMS PCT assay, BRAHMS PCT Sensitive Kryptor assay, Elecsys BRAHMS PCT assay, LIAISON BRAHMS PCT assay and VIDAS BRAHMS PCT assay); Diagnostics guidance [DG18].2015.\n\nNational Institute for Health and Care Excellence: Antimicrobial stewardship: systems and processes for effective antimicrobial medicine use (NG15). NICE Guidance. 2015." }	[ { "id": "97615", "date": "10 Nov 2021", "name": "Paul J. Gibson", "expertise": [ "Reviewer Expertise I am a clinical pediatric oncologist with research experience in supportive care. I cannot however", "confidently suggest all statistical methods quoted in the paper are appropriate." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments:\nThis is a nice small study that tackles an interesting and still unanswered question with regards to the utility of biomarkers in the risk stratification of Pediatric Febrile Neutropenia. However, it appears there were challenges in recruitment and sample collection that are not specifically outlined. It may be helpful to describe those challenges to guide future efforts.\n\nAbstract:\nConclusions: \"Repeated assessments over time may be of value\". I'm not convinced that the body of the paper nor the discussion justify this statement based on the results of this study. While other studies are referenced that may point to the utility of serial assessments, the concept is not substantially discussed in this paper.\nIntroduction:\nThe second paragraph mentions interest in newer biomarkers but fails to address CRP until it used as a single example in the 3rd paragraph. I suggest the authors make earlier reference to this 'older' biomarker and its role (or lack thereof) in risk stratification and highlight why PCT IL-6 and IL-8 may be preferable.\n\nMethods:\n\"Using this unselective approach, with treatment unaffected by the results of the biomarker analyses, we minimized the biases which can arise through cherry-picking of patients and altering medical treatment on the basis of the test under evaluation (selection and incorporation biases)\". This sentence may be more succinctly stated. such as \"PCT, IL-6 and IL-8 values were not provided to clinicians to avoid bias\"\nAs Table 1 states results, it is more appropriate in the results section.\nThe methods reference power calculations suggesting ~400 episodes were required. but only 43 were captured, yet there is no explanation for planned duration or reason for termination of the study.\n\nResults\nData was collected on 65 patients, yet only 31 have had biomarkers taken? Why? Did they refuse collection? Did they present outside of times feasible for biomarker collection? The Discussion references technical and administrative challenges, but maybe expand what you mean?\n\nYou may consider showing graphically the changes in biomarkers in the 5 SMC patients.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7766", "date": "03 Feb 2022", "name": "Bob Phillips", "role": "Author Response", "response": "\"General Comments: This is a nice small study that tackles an interesting and still unanswered question with regards to the utility of biomarkers in the risk stratification of Pediatric Febrile Neutropenia. However, it appears there were challenges in recruitment and sample collection that are not specifically outlined. It may be helpful to describe those challenges to guide future efforts.\" This is an extremely good point, and paragraph 3 has been added to the Discussion. \"Abstract: Conclusions: \"Repeated assessments over time may be of value\". I'm not convinced that the body of the paper nor the discussion justify this statement based on the results of this study. While other studies are referenced that may point to the utility of serial assessments, the concept is not substantially discussed in this paper.\" This is also reasonable – the few data there are congruent but an overstatement from this report. Modified. \"Introduction: The second paragraph mentions interest in newer biomarkers but fails to address CRP until it used as a single example in the 3rd paragraph. I suggest the authors make earlier reference to this 'older' biomarker and its role (or lack thereof) in risk stratification and highlight why PCT IL-6 and IL-8 may be preferable. \" A worthwhile negative to explicitly state. Added to into paragraph 3 to explain why the newer ones are possibly better. \"Methods: \"Using this unselective approach, with treatment unaffected by the results of the biomarker analyses, we minimized the biases which can arise through cherry-picking of patients and altering medical treatment on the basis of the test under evaluation (selection and incorporation biases)\". This sentence may be more succinctly stated. such as \"PCT, IL-6 and IL-8 values were not provided to clinicians to avoid bias\" This is definitely true, but I’d really like to keep the longer explanation. As a tutor of critical appraisal, I have found the word “bias” is often thrown about as a correct answer, but more knowing it’s the code to use than the meaning of it. Additionally, it conforms more strongly to the STROBE checkpoint 9. As Table 1 states results, it is more appropriate in the results section. Fair. This is now a supplementary Table, and Table 1 is within the results. The methods reference power calculations suggesting ~400 episodes were required. but only 43 were captured, yet there is no explanation for planned duration or reason for termination of the study. Again, reasonable request for the missing information. Added in Methods and Discussion. \"Results: Data was collected on 65 patients, yet only 31 have had biomarkers taken? Why? Did they refuse collection? Did they present outside of times feasible for biomarker collection? The Discussion references technical and administrative challenges, but maybe expand what you mean? \" Agreed – discussion paragraph added. Final Comment: You may consider showing graphically the changes in biomarkers in the 5 SMC patients. It is definitely worth considering. Given the very small numbers and the lack of real value of the work in serial measurements arising from this report, as correctly emphasised earlier, adding a graph might well over-egg this part of the pudding again and is probably better left text-based." } ] }, { "id": "100188", "date": "21 Jan 2022", "name": "Rejin Kebudi", "expertise": [ "Reviewer Expertise Pediatric oncology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript Prospective cohort study of the predictive value of inflammatory biomarkers over clinical variables in children and young people with cancer presenting with fever and neutropenia investigates the role of PCT, IL6, IL8 at first day and serial monitoring in paediatric febrile neutropenic episodes.\nThe unselective approach for the biomarkers (the inflammatory biomarker results except for CRP were masked from the treating team) minimized the biases for medical treatment. Did the results of CRP (which the medical team could see) effect treatment decisions?\nThere were 43 episodes and 31 patients, only 31 episodes had first day data. Despite that, the biomarker levels concurred better with the physician assessment of severe clinical illness, however, this was not statistically significant. The conclusion was that at presentation of febrile neutropenic episodes, serum biomarkers of inflammation do not improve the risk stratification, repeated assessment over time may be of value.\nWere there any differences in patients who had acute leukemia vs solid tumors? Were all of the febrile neutropenic episodes, in which biomarkers were investigated, high risk? If there were both high risk and low risk episodes, did the biomarkers differ within the risk groups?\nIn table 3 the mean inflammatory biomarker values in severe clinical appearance are numerically much higher than the non-severe ones; what was the range of each biomarker in the severe and non-severe group. Do the authors define the severe clinical appearance (table 3) the same as SMC?\nAlthough the use of these biomarkers were not statistically significant in initial stratification, the evaluation of a larger cohort with higher number of SMC, may give more information on the value of the initial inflammatory biomarkers for predicting a SMC or sepsis. A larger cohort may also give more information for the value of the use of serial biomarker monitoring and which one/ones to use cost-effectively. The discussion may include some of these concerns.\nDo see this for further 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? Yes\n\nAre 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": "7767", "date": "03 Feb 2022", "name": "Bob Phillips", "role": "Author Response", "response": "There are a number of interesting questions raised in the review: Were there any differences in patients who had acute leukemia vs solid tumors? This is a very fair question, and no, there were no qualitative differences in patient type. Given the small numbers, multiple analyses for variation were felt to be unwise and only the highest level (risk-grouping and biomarkers) were undertaken. Were all of the febrile neutropenic episodes, in which biomarkers were investigated, high risk? If there were both high risk and low risk episodes, did the biomarkers differ within the risk groups? The risk groups are shown in Extended Data Table 2; they included all levels of risk. In table 3 the mean inflammatory biomarker values in severe clinical appearance are numerically much higher than the non-severe ones; what was the range of each biomarker in the severe and non-severe group. This is a good question and Table 3 has been modified to add the (range) as well as the mean values. Do the authors define the severe clinical appearance (table 3) the same as SMC? No – severe clinical appearance and significant medical complication are defined differently. SMC is defined in the fifth methods paragraph, “severe clinical appearance” is the gestalt impression of the examining physician." } ] } ]	1	https://f1000research.com/articles/10-1070
https://f1000research.com/articles/10-47/v1	26 Jan 21	{ "type": "Research Article", "title": "Human Proteinase 3, an important autoantigen of c-ANCA associated vasculitis, shares cross-reactive epitopes with serine protease allergens from mites: an in silico analysis", "authors": [ "Emiro Buendía", "Múnera Marlon", "Orlando Parra", "María Sánchez", "Andrés Sánchez", "Jorge Sánchez", "Diego Viasus", "Múnera Marlon", "Orlando Parra", "María Sánchez", "Andrés Sánchez", "Jorge Sánchez", "Diego Viasus" ], "abstract": "Background: In autoimmune vasculitis, autoantibodies to Human Proteinase 3 (PR3), a human serine protease, seems to have a role on the inception of c-ANCA associated vasculitis. The origin of this autoreactive response remains unclear. However, for several autoreactive responses, molecular mimicry between environmental antigens and human proteins is key to trigger autoantibodies and finally autoimmunity manifestations. Considering that PR3 is a serine protease and house dust mite (HDM) group 3 allergens share this biochemical activity, the aim of this study was to identify cross-reactive epitopes between serine proteases from human and mites using an in silico approach. Methods: Multi alignment among amino acid sequences of PR3 and HDM group 3 allergens was performed to explore identity and structural homology. ElliPro and BepiPred in silico tools were used to predict B and T cell epitopes. Consurf tool was used to conduct identification of conserved regions in serine proteases family. Results: PR3 and HDM group 3 allergens shared moderate identity and structural homology (root mean square deviation < 1). One B cell cross reactive epitope among serine proteases was identified (29I, 30V, 31G, 32G, 34E, 36K, 37A, 38L, 39A and 54C) and two T cell epitopes. Conclusions: PR3 have structural homology and share cross reactive epitopes with HDM group 3 allergens.", "keywords": [ "Serine proteases", "human proteinase 3", "house dust mites group 3 allergens", "ANCA associated vasculitis", "sequence homology", "T and B cell epitopes", "cross-reactivity", "epitope modelling" ], "content": "Introduction\n\nAnti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a life-threatening autoimmune disease affecting small vessels, compromising the respiratory mucosa, skin, lung, and the kidney1. Different diseases are present in this group of small vessel vasculitis; granulomatosis with polyangiitis, microscopic polyangiitis, kidney-limited vasculitis and Eosinophilic granulomatosis with polyangiitis. In patients suffering AAV, autoantibody binding to the Human Proteinase 3 protein (PR3) expressed on the neutrophil surface may activate its degranulation, eliciting tissue damage in small vessels and their irrigated organs. Also, proinflammatory effector T cells have been implicated in vasculitis pathogenesis2, but a specific PR3 T cell epitope has not been reported in AAV patients3. PR3 is a serine protease physiologically expressed in human neutrophils. Due to its enzymatic activity it degrades various intercellular gap-junction proteins and collagen and may play a role in neutrophil transendothelial migration. Besides, this protein is an important autoantigen in AAV, and sera from patients with severe and relapsing forms of the disease can bind it in IgG ELISA assays4–6. Although, a cause-effect relationship between PR3-autoantibodies and vasculitis is not clearly defined, animal models supports a pathogenic role7,8 and they may be involved in disease inception, progression and severity1.\n\nEnvironmental exposures, specially to microbial components mimicking self-antigens have been proposed as triggers of autoimmunity9,10. Also, in AAV, it have been proposed that an endogenous immune response to a complementary protein to PR3 autoantigen could be implicated in disease inception, and this antisense protein harbors homology to various bacterial peptides11. PR3 crystal structure has been elucidated, and various epitopes are recognized by patients suffering AAV, however its cross-reactivity with environmental antigens is poorly studied12–14.\n\nPrevious studies have shown that specific IgE to some self-proteins have been identified in autoimmune and allergic diseases like lupus, urticaria, dermatitis, allergic pulmonary aspergillosis and have a strong association with disease activity15–18. Some allergens can cross-react with human proteins and participates in autoimmunity inception in pemphigus vulgaris by a “hit-and-run” mechanism, opening the theoretical possibility for a similar mechanism to occur in another autoimmune diseases such as AAV19–22.\n\nIn the tropics, house dust mites (HDM) are important ubiquitous allergen sources and exposure is perennial, increasing the possibilities of exposure in the general population23, and IgE sensitization to their components24,25. Sensitization to HDM group 3 allergens is common26, they harbor serine protease activity and conserved structural homology27, making them potential PR3 cross reactive antigens, however it has not been explored before. Here, we show in silico data suggesting cross-reactivity and epitope sharing between PR3 and HDM group 3 allergens.\n\n\nMethods\n\nThe amino acid sequence from the human PR3 (Uniprot accession: P24158) was used as query to perform a search for serine protease homologous reported in allergenic sources: Dermatophagoides pteronyssinus (Der p 3: Accession number P39675), Blomia tropicalis (Blo t 3: A1KXI1), Glycyphagus domesticus (Gly d 3: Q1M2M8), Lepidoglyphus destructor (Led p 3: Q1M2L7) and Tyrophagus putrescentiae (Tyr p 3: C6ZDB5) with the PSI-BLAST tool. Parameters were set as default.\n\nIdentity among all allergenic sequences homologous to PR3 was analyzed by Jalview tool2.11.028. First, all allergens and human PR3 codes were used as inputs in Jalview tool. Second, T coffee tool was chosen to assess alignment. Third, alignment was displayed as identity percent.\n\nThe 3D model of Der p 3, a serine protease of Dermatophagoides pteronyssinus was generated by homology using the SWISS-MODEL server. The 3D model of Der p 3 was loaded into ProSA-web server29, which was used to analyze its quality.\n\nThe model was refined in DeepView v4.1 (energy minimization and rotamer replacements). Its quality was evaluated by several tools, including Ramachandran graphs, WHATIF, QMEAN4 index, and energy values (GROMOS96 force field).\n\nThree-dimensional structures (PDB: 1FUJ) of the human serine protease was retrieved from the Protein Data Bank. Cartoon model was created using Pymol software v2.4. Root median square deviation (RMSD) value between Der p 3 and PR3 was calculated using Chimera software v1.030.\n\nElliPro v3.0 and BepiPred v2.0 tools were used to predict B and T cell epitopes on Der p 331,32. With ElliPro, the 3D structure of Der p 3 was used to predict epitopes. Minimum score and maximum distance (Angstrom) were set to 0.5 and 6, respectively. Epitopes with high conserved rates were visualized on 3D model. For prediction using BepiPred, amino acid sequence of Der p 3 was used as input.\n\nThe 3D structure of Der p 3 was submitted to the ConSurf server to generate evolutionarily related conservation scores to help to identify functional regions in the proteins. HMMER algorithm, 1 iteration, E-value cutoff (0.0001) and UNIREF-90 database was set as default to generate multiple alignment, previous to evolutive analysis. All amino acid sequences in FASTA format were used.\n\n\nResults\n\nBLAST search identified various serine protease family members from HDM as homologous. Here, allergens such as: Der p 3, Blo t 3, Gly d 3, Led p 3 and Tyr p 3 shared an identity of 45% among them according to multiple sequence alignment (Figure 1). Among the members of HDM group 3 allergens, an identity till 41% was reported (Table 1), and a highly conserved region between residues 40 to 90 was found. When identity between PR3 and each allergen used in study was analyzed, a moderate level of identity was found (30%) (Table 1).\n\nAn identity of 45% in their amino acid sequences was found.\n\nAll comparisons of PR3 with HDM group 3 allergens showed a moderate identity.\n\nA structural model of Der p 3 was obtained by homology modelling using the 3D structure of PR3 reported in PDB database. According to modelling, Der p 3 tertiary structure exhibited a typical fold of serine protease family, conformed by four α-helixes and fifteen β-strands with structural homology with PR3 (RMSD = 0.8) (Figure 2).\n\n(A) PR3; (B) Der p 3. According to modelling by homology, Der p 3 exhibited a typical fold of serine protease family. (C) Overlapping of 3D model of PR3 and Der p 3. RMSD value of 0.8 is reported, suggesting structural homology.\n\nUsing ElliPro and BepiPred servers, a cross reactive B cell epitope was predicted on all serine protease used in this study. This epitope is formed by ten residues and is on loop region, spanning amino acids 29 and 39 with a surface area of 470 Å. Conservative analysis indicated that the antigenic region predicted was highly conserved in the serine proteases (Figure 3). According to ConSurf analysis, the region covering the cross-reactive epitope is conserved among the serine protease family (Figure 4). T cell epitope prediction identified at least two epitopes with potential cross-reactivity among all sequence analyzed. Both epitopes are located on β strands, first epitope span 45 to 59 region (ISLQSSSHFCGGTIL) and the second, the 63 to 77 region (WILTAAHCVAGQTAS) (Figure 5; Table 2).\n\n(A) Surface model of Der p 3, showing area occupied by B cell epitope predicted as cross reactive. (B) Cartoon model showing location of epitope on tridimensional structure. It can be appreciated that the predicted epitope is on a loop spanning residues 29 to 39.\n\n(A and C) Cartoon models showing the conserved region among serine proteases. (B and D) Surface models showing conserved region among serine proteases.\n\nIt can be appreciated that predicted epitopes are in a continuous β strands (blue and magenta).\n\nBoth are conserved among PR3 and HDM group 3 allergens.\n\n\nDiscussion\n\nIn this study we found that PR3 and HDM group 3 serine protease allergens have conserved identity and homology. Also, for the first time, we predicted various T and B cell cross reactive epitopes between them through an in silico approach. PR3 is an important autoantigen in small vessel vasculitis and it seems to participate in disease inception, progression and severity1. Our results have potential implications for the understanding of autoreactive response in AAV and open the possibility for a new environmental trigger of the autoreactive response in AAV.\n\nIn AAV, it have been proposed that autoantibodies directed to a complementary protein to PR3 autoantigen could be implicated in disease inception, and this antisense protein harbors homology to various bacterial peptides11, a theory named autoantigen complementarity33. However, in epidemiological studies, autoantigen complementarity hypothesis testing have showed conflicting results, since sera from some patients suffering AAV do not recognize complementary PR3, but others do recognize it34–36. Also, molecular mimicry of PR3 protein by infectious microorganism components have been proposed as a possible environmental trigger of the disease based on the reports of infections preceding the manifestations of vasculitis10,37–39, but a cross reactive antigen have not been reported yet.\n\nIn their seminal publication, Pendergraft et al. ran a BLAST query to find homologues of PR3 protein in microbial or fungal microorganisms, and did not find matching sequences at that time11. However, they did not include Arachnida or other environmental sources of cross-reactivity. In our analysis we found matching PR3 protein sequences with various HDM group 3 serine protease allergens, and at least theoretically this finding could have many implications for the understanding of inception and even diagnosis of autoreactive response in AAV. Recently, Qian et al have shown that some allergens can cross-react with human proteins19 and participate in autoimmunity inception in pemphigus vulgaris by a “hit-and-run” mechanism22, opening the theoretical possibility for a similar mechanism to occur in another autoimmune disease such as AAV. Similarly, in atopic dermatitis, Valenta and collaborators observed that some patients with severe conditions of the disease, had IgE directed to the profilin of the Betula verrucosa, but also to the human homologue40.\n\nIn the tropics, HDM are important ubiquitous sources of protease allergens. Exposure is perennial, increasing the possibilities of exposure and IgE sensitization to their components in the general population23–25. Sensitization to HDM group 3 allergens is common26, and they harbor serine protease activity27, a characteristic that make them highly allergenic, conserved structural homology that make them highly immunogenic41,42 and suitable for epitope spreading43. In this context, “hit-and-run” and epitope spreading set framework mechanisms for environmental allergens with homology to autoantigens to potentially participate in the development of autoimmunity. We speculate that HDM group 3 allergens harbor two characteristics that make them suitable candidates for environmental triggering of AAV: their proteolytic activity that, as other protease allergens, set a tissue damaging microenvironment during antigen recognition41; and molecular homology-epitope sharing with human PR3, that would elicit B cell autoantibody production and autoreactive T cell receptor generation. In conclusion, we found that PR3 and HDM group 3 serine protease allergens have conserved identity, and for the first time we predicted cross reactive epitopes between them through an in silico approach.\n\n\nData availability\n\nUniProtKB: PRTN3_HUMAN, Accession number P24158: https://www.uniprot.org/uniprot/P24158\n\nProtein Data Bank: PR3 (MYELOBLASTIN), Accession number 1FUJ: https://www.rcsb.org/structure/1FUJ\n\nUniProtKB: Mite allergen Der p 3, Accession number P39675: https://www.uniprot.org/uniprot/P39675\n\nUniProtKB: Trypsin Blo t 3, Accession number A1KXI1: https://www.uniprot.org/uniprot/A1KXI1\n\nUniProtKB: Gly d 3, Accession number Q1M2M8: https://www.uniprot.org/uniprot/Q1M2M8\n\nUniProtKB: Allergen Lep d 3, Accession number Q1M2L7: https://www.uniprot.org/uniprot/Q1M2L7\n\nUniProtKB: Trypsin Tyr p 3.0101, Accession number C6ZDB5: https://www.uniprot.org/uniprot/C6ZDB5", "appendix": "References\n\nJennette JC, Falk RJ: Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. 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PubMed Abstract \| Publisher Full Text\n\nCayrol C, Duval A, Schmitt P, et al.: Environmental allergens induce allergic inflammation through proteolytic maturation of IL-33. Nat Immunol. 2018; 19(4): 375–85. PubMed Abstract \| Publisher Full Text\n\nWesternberg L, Schulten V, Greenbaum JA, et al.: T-cell epitope conservation across allergen species is a major determinant of immunogenicity. J Allergy Clin Immunol. 2016; 138(2): 571–8.e7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurastero SE: T-cell receptor-mediated cross-reactivity to different allergens is driven by recognition of homologous, phylogenetically conserved epitopes. J Allergy Clin Immunol. 2016; 138(4): 1237. PubMed Abstract \| Publisher Full Text" }	[ { "id": "90330", "date": "09 Aug 2021", "name": "Chandrabose Selvaraj", "expertise": [ "Reviewer Expertise Immunoinformatics", "Computational Biology", "Molecular 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\nReviewer Recommendation: Major Revision\nPrimary check on this manuscript for language refinement is mandatory as I see that it is not satisfactory, and moreover, there are more typographical errors. I suggest the authors go with language and grammar checks.\n\nThe following sentence seems incomplete and difficult to understand, reframe it “Here, allergens such as: Der p 3, Blo t 3, Gly d 3, Led p 3 and Tyr p 3 shared an identity of 45% among them according to multiple sequence alignment\"\n\nWhy have authors selected PR-3 for the homologous evaluation study? Since the 3-D structure is available in the PDB database?\n\nWhat basis authors have selected allergenic sources such as Dermatophagoides pteronyssinus (Der p 3: Accession number P39675), Blomia tropicalis (Blo t 3: A1KXI1), Glycyphagus domesticus (Gly d 3: Q1M2M8), Lepidoglyphus destructor (Led p 3: Q1M2L7) and Tyrophagus putrescentiae (Tyr p 3:C6ZDB5) in this study? In BLAST it did not show the above mentioned allergens.\n\nDid authors find the sequence similarity analysis of target sequence Der p 3 with its template; if so provide that information more in detail.\n\nSwiss-Modeler does not show the 1FUJ as template in template searching, and then what basis authors have checked RMSD calculation between the modeled structure and IFUJ as template?\n\nIn methodology part authors have mentioned that the modeled structure was subjected to validation, but lack results and interpretation. Provide more in detail.\n\nProvide abbreviation for AAV\n\nIs the residues present in the epitope region responsible for the antigenic effect?\n\nIn epitope prediction, the author only provides the sequence and length of the epitopes, if the author provides the information on score value, identity and whether it is present in any domain or motif it could be more informative.\n\nIs the work 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?\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": "7564", "date": "03 Feb 2022", "name": "Emiro Buendía", "role": "Author Response", "response": "Emiro Buendía, Division of Health Sciences, Universidad del Norte, Barranquilla, Colombia. Dear reviewer, thanks for the reading of the paper, we have taken note of all your comments, questions and suggestions. Our responses are bellow: 1. Reviewer: Primary check on this manuscript for language refinement is mandatory as I see that it is not satisfactory, and moreover, there are more typographical errors. I suggest the authors go with language and grammar checks. Answer: We appreciate your suggestion. The manuscript was revised and refined by a language style corrector. 2. Reviewer: The following sentence seems incomplete and difficult to understand, reframe it “Here, allergens such as: Der p 3, Blo t 3, Gly d 3, Led p 3 and Tyr p 3 shared an identity of 45% among them according to multiple sequence alignment\" Answer: We appreciate your suggestion. A new sentence was added: “Multiple sequence alignment showed that Der p 3, Blo t 3, Gly d 3, Led p 3 and Tyr p 3 allergens shared 45% of identity in their aminoacid sequences with PR3. The most conserved region is located between residues 53 to 75, indicating the existence of molecular mimicry.” 3. Reviewer: Why have authors selected PR-3 for the homologous evaluation study? Since the 3-D structure is available in the PDB database? Answer: We appreciate your question. We selected PR3 for the study because it is the most important autoantigen in ANCA + vasculitis. Since, this human protein is a serine protease, in the sequence alignment analysis we seek for other serine proteases in mites, because it is a previously described environmentally ubiquitous serine protease source. In our study we selected the 3D model of PR3 from the Protein Data Bank as mentioned in your observation because its structure was already deciphered, the structural homology analysis was done keeping in mind that its serine protease folding could make more possible for us to model homologous serine in mites. 4. Reviewer: What basis authors have selected allergenic sources such as Dermatophagoides pteronyssinus (Der p 3: Accession number P39675), Blomia tropicalis (Blo t 3: A1KXI1), Glycyphagus domesticus (Gly d 3: Q1M2M8), Lepidoglyphus destructor (Led p 3: Q1M2L7) and Tyrophagus putrescentiae (Tyr p 3:C6ZDB5) in this study? In BLAST it did not show the above-mentioned allergens. Answer: We appreciate your question. These allergens were chosen on the basis that they belong to the serine protease family, and our hypothesis was that the sensitization to these allergens is cross-reactive with the PR3 autoantigen. So, search was limited using terms: American house dust mite (taxid:6954). In this way, results were only about mites, an important allergenic source. 5. Reviewer: Did authors find the sequence similarity analysis of target sequence Der p 3 with its template; if so provide that information more in detail. Answer: We appreciate your question. Yes, the binary alignment showed 33% identity between the template amino acid sequences, and Der p 3. An image of the alignment was uploaded in the new manuscript version. 6. Reviewer: Swiss-Modeler does not show the 1FUJ as template in template searching, and then what basis authors have checked RMSD calculation between the modeled structure and IFUJ as template? Answer: We appreciate your question. For modelling the mite allergen Der p 3 structure, we used the zymogen catalytic region of MASP-2, a lectin binding mannose from human (PDB: 1zjk f), as a template because 1FUJ code was not listed in Swiss-Modeler query results for this protein. Next we calculated the RMSD based on the predicted structure of the mite Der p3 protease and the previously resolved structure of the human PR3. The following sentence was improved to answer your question in the method section of the paper (Construction of 3D model): “The 3D model of Der p 3, a serine protease of Dermatophagoides pteronyssinus was generated by homology in the SWISS-MODEL server using the zymogen catalytic region of human MASP-2 (PDB: 1zjk f) as a template. The 3D model of Der p 3 was loaded into the ProSA-web server 29 , which was used to analyze its quality”. 7. Reviewer: In methodology part authors have mentioned that the modeled structure was subjected to validation, but lack results and interpretation. Provide more in detail. Answer: We appreciate your suggestion. We added the next sentence in the method section (Construction of 3D model): For the validation of the Der p 3 structure we used the Minimize Structure option in the UCSF Chimera software, a procedure that adjust the energy (entropy) of the model. 8. Reviewer: Provide abbreviation for AAV. Answer: We appreciate your suggestion. ANCA Associated Vasculitis (AAV) was included in the corrected paper text. 9. Reviewer: Is the residues present in the epitope region responsible for the antigenic effect? Answer: We appreciate your question. We think that the residues found in the epitope region are involved in the cross reactivity between the studied serine protease and hypothesized have a role in autoimmunity inception. But this are preliminary results awaiting experimental validation. 10. Reviewer: In epitope prediction, the author only provides the sequence and length of the epitopes, if the author provides the information on score value, identity and whether it is present in any domain or motif it could be more informative. Answer: We appreciate your suggestion. The B epitope is part of the N-terminal region of PR3 and is not within any domain of the protein. The T epitope spanned between residues 45-59 is located on the first and second beta lamina. While the second epitope T (63-77) is located in the third beta sheet, and first alpha helix in the protein structure. In the result section (T and B cell cross-reactive epitopes were predicted between HDM group 3 allergens and PR3), two sentences were improved to consider your suggestion." } ] } ]	1	https://f1000research.com/articles/10-47
https://f1000research.com/articles/9-1230/v1	13 Oct 20	{ "type": "Research Article", "title": "Career planning courses increase career readiness of graduate and postdoctoral trainees", "authors": [ "Rebekah L. Layton", "V. Scott H. Solberg", "Arthee E. Jahangir", "Joshua D. Hall", "Christine A. Ponder", "Keith J. Micoli", "Nathan L. Vanderford", "V. Scott H. Solberg", "Arthee E. Jahangir", "Joshua D. Hall", "Christine A. Ponder" ], "abstract": "Background: Given national calls for intentional career development during graduate and post-graduate scientific training, this study assessed career readiness development within the context of academic career courses. The current study evaluated the effects of academic career courses offered at two institutions that were specifically designed to increase career awareness, interest, and career-related confidence among doctoral students and postdoctoral fellows. Methods: Participants enrolled in a career course at trainees’ respective academic institutions and responded to pre- and post-course surveys (n=32, n=148). The paper offers a thematic analysis of each of the two courses using an individualized learning plan career development framework and describes the results of their respective pretest-posttest evaluations which indicated increases in career readiness. Results: Though the format and content provided in each course varied, participation was associated with increases in career readiness. Participants reported increased career-awareness including a greater familiarity with different types of careers overall. Furthermore, interest in tenure track faculty careers increased in both samples, which may assuage fears that exposure to diverse career pathways could reduce interest in academic careers. Transferrable skills, including career planning and awareness also significantly increased. Course participants reported an increase in the number and type of mentors they interacted with beyond their principal faculty mentor (other faculty, professional PhDs, peers, and administrative staff). Conclusions: Findings provide supporting evidence for the benefits of implementing structured career development efforts during PhD training; even with varying content, delivery methods, and instructor type, both academic career courses led to significant gains in career awareness and readiness. Successful development and delivery of academic career courses, with a focus on career planning skills, suggest that institutions can utilize these and are an effective way to prepare PhDs for their transition from training positions into careers.", "keywords": [ "Graduate education", "professional development", "career choice" ], "content": "Introduction\n\nTraditionally, doctoral and postdoctoral training employs a model by which graduate students and postdoctoral fellows work under the guidance and mentorship of experienced faculty members to prepare for similar careers in academia. Especially in the biomedical sciences, this often includes mentorship from a primary faculty advisor in expectation of one day becoming a principal investigator as well. Although this trend is evident across disciplines, in recent decades and in the biomedical sciences in particular, the number of doctoral trainees has grown more quickly than the number of faculty positions available (NIH, 2012; Stephan, 2012). Even after years of postdoctoral experience, two-thirds of biomedical PhD graduates are finding employment in non-tenure track faculty positions or nonacademic settings (Kahn & Ginther 2017; NIH, 2012). Given the relative scarcity of tenure-track opportunities, the National Institutes of Health (NIH) and other funders have encouraged the biomedical training system to provide career development support for early career biomedical scientists to facilitate exploration of non-academic career pathways and to build professional skills (Denecke et al., 2017; NIH, 2012). Despite this, the traditional training model has remained fairly consistent in preparing doctoral and postdoctoral trainees for academic careers despite the fact that the career landscape offers a wide range of opportunities in other sectors.\n\nBoth empirical and anecdotal evidence also suggest that during doctoral training many students lose interest in pursuing academic careers (Fuhrmann et al., 2011b; Gibbs & Griffin, 2013; Layton et al., 2016; Roach & Sauermann, 2010; Sauermann & Roach 2012). As a result of these trends, recent efforts to improve biomedical research training has included calls for providing career development opportunities that promote awareness of the variety of careers that align with the doctoral-level skills and competencies associated with receiving a PhD in a biomedical field (Alberts et al., 2014; Fuhrmann, 2016; Hitchcock et al., 2017; McDowell et al., 2014; Pickett et al., 2015).\n\nTo encourage these efforts, NIH established the Broadening Experiences in Scientific Training (NIH BEST; Lenzi et al., 2020; Meyers et al., 2016) and the National Science Foundation established Non-Academic Research Internships for Graduate Students (INTERN; Tornow et al., 2018). The motivation of national funding agencies such as these to prioritize creation of career development programs and services for biomedical doctoral and postdoctoral trainees is based on evidence that graduates lack training and exposure to options that reflect the wide variety of labor market opportunities associated with the diverse biomedical industry in the United States, especially among those who remain for an extended period in post-doctoral positions (NIH, 2012). Additional data suggests that even with increased career familiarity, career goal clarity remains low for both graduate trainees and postdoctoral fellows (Gibbs et al., 2015). To date, the design and implementation of career development programs and services in biomedical programs is only recently beginning to become broadly accepted beginning (i.e., https://careerdevelopment.aaas.org/; Denecke et al., 2017; Mathur et al., 2018; Schnoes et al., 2018). Furthermore, while career courses have been widely utilized and studied in undergraduate populations (Reese & Miller, 2006), integration of career training into graduate coursework and curricula is relatively rare (Fuhrmann, 2016) and there is a paucity of research evaluating its effectiveness at the doctoral and postdoctoral levels.\n\nThis study reports on the initial effort of two institutions to offer doctoral career development courses in the form of academic credit-bearing courses, each designed independently, while sharing a common goal of supporting exploration of the many career opportunities that can be found outside of academia. The purpose of this study was to: (a) use a career development framework from which to conduct a thematic analysis of the course content, and (b) analyze course evaluations to assess whether adding a career development course shows promise in facilitating doctoral and postdoctoral trainees’ career exploration skills, as well as consideration of alternative career opportunities beyond academia.\n\n\nCareer Course Thematic Analysis\n\nThe first career course examined in this study was created and implemented at a large public university in the south-central region of the United States as an academic career-development course that was cross-listed as “Preparing Future Professionals” and “Preparing Science Professionals” (PSP; see Norman-Burgdolf & Vanderford, 2016 and Vanderford, 2017 for more about the development of the course). The second course was created by a large private university in the northeastern region of the United States as an academic career-development course entitled “Hope is Not a Plan (HINAP): Taking Charge of Your Science Career.” The first course (subsequently referred to as “PSP”) is taught by a faculty member, while the second course (subsequently referred to as “HINAP”) is taught by professional development staff.\n\nIn conducting a thematic analysis of the course content, the syllabi content was examined in relation to how course participants engage in three career development skill domains (Solberg, 2019; Solberg et al., 2018): self-exploration, career exploration, and career management and planning. Program activities that develop self-exploration skills are those designed to help participants become aware of their talent, skills, interests, and values. Activities that develop career exploration skills are those that help participants identify: (a) how their talent and skills transfer into a wide range of career opportunities, (b) emerging labor market opportunities and the competencies needed to pursue them, and (c) career and life goals. Career management and planning activities involve: (a) job search skills such as informational interviewing, interview skills, and resume preparation, as well as (b) plans for acquiring the additional technical and human relation skills needed to successfully pursue their career and life goals. This framework is a variation from the traditional themes of “career awareness,” “career exploration,” and “career preparation/career immersion” (e.g., Colorado, Massachusetts) derived from a multi-method, multi-study of individualized learning plans (ILPs; Solberg et al., 2014). Currently ILPs have been adopted by a number of states (U.S. Department of Labor, ODEP, https://www.dol.gov/agencies/odep/topics/individualized-learning-plan/map), and many of the states have moved to adopting this revised career development framework (e.g., Solberg, 2019). This revised framework differs from the traditional focus on career awareness, exploration, and preparation in two fundamental ways. First, the revised framework focuses on providing evidence-based activities that result in an individual acquiring career development “skills.” Traditionally, career development programs focused primarily on providing activities but did not emphasize the idea that these activities should result in positive development outcomes such as becoming proactive and self-directed with respect to engaging in future career development efforts (Solberg & Ali, 2017). The second departure from the traditional model involves replacing “career awareness” with self-exploration. Career awareness activities are most strongly associated with a career decision-making paradigm whereby the process begins with completing a career interest inventory with the purpose of narrowing career options that best match their personality type (Hartung et al., 2015). In response to new advances in career theory such as Life Design (Savickas, 2016; Savickas et al., 2009) and increasing disruptions in the world of work brought about by technology and globalization (Schwab, 2019), made even more relevant by current job market trends related to the global response to COVID-19 (Mathur, 2020), the focus on self-exploration skills has been used to bring more focus on skill-building activities that help individuals learn how to identify their skills, interests and values (including in graduate education; Vanderford et al., 2018a & Vanderford et al., 2018b) and examine whether and how these transfer into a wide range or career opportunities (Solberg, 2019).\n\nTable 1 summarizes this syllabus content of each course in relation to the revised career development framework described above. In many ways, the pedagogy in PSP is consistent with Life Design (Savickas, 2016; Savickas et al., 2009) and the integration of constructivist models of career assessment and counseling (Brott, 2004; Schultheiss, 2005). For example, the course emphasizes the use of classroom discussions and written career narratives that engage participants in an idiographic assessment of their transferable skills (i.e., relies on examination of unique and subjective experiences), and the use of classroom discussions to further explore career goals and develop plans to pursue those goals. Alternatively, HINAP uses a more traditional nomothetic assessment and structured career planning strategy. For example, to begin the course, HINAP participants complete an online skills assessment that matches the participants results to a range of career pathways and uses the online system to complete a structured set of career development planning activities.\n\nComparison of PSP Course and the HINAP Course using an ILP Framework identifies two common areas of career development themes identified (career exploration skills; and career management and planning skills).\n\nWith respect to self-exploration skills, PSP provides opportunities for participants to reflect on their talent, skills, and interests. For PSP, the goal is for course participants to begin by reflecting on their current transferable skills and then to develop career exploration skills through exposure to career paths that align with those skills. Based on the ILP career development framework, rather than incorporating self-exploration as a self-discovery process, HINAP’s syllabus relied instead on the use of a skills assessment strategy; as result, this activity was classified under career exploration.\n\nWith respect to developing career exploration skills, both PSP and HINAP incorporated role models and informational interviews as a strategy for expanding participants awareness of how their transferable skills align to a wide range of careers. PSP used this to help course participants reflect on how their transferable skills connect to different career pathways and to encourage establishment of career goals. Alternatively, HINAP directed course participants to complete an online individual development plan (IDP for PhD scientists; MyIDP) provided by Science Careers and that was specifically designed for the biomedical field (Fuhrman et al., 2011a; Hobin et al., 2012). MyIDP is a free-access system (https://myidp.sciencecareers.org/) sponsored by the American Association for the Advancement of Science (AAAS) that helps users explore to what extent their skills and interests match to 20 scientific career paths (Table 2). MyIDP also helps users identify short-term goals to further develop and expand one’s skills and advance in one’s career. Using the MyIDP system features, career exploration activities include a skills assessment and career pathway matching exercise, identification of long-term career advancement goals, and selection of potential career pathways of interest for further exploration.\n\nSample biomedical career pathway definitions drawn from MyIDP are displayed to illustrate the diverse career options available to graduate and postdoctoral trainees during career exploration.\n\nWith respect to career management and planning skills, both PSP and HINAP emphasize a number of job search skills including preparing resumes and cover letters and learning where to look for viable job opportunities. Both syllabi focus goal setting, with HINAP additionally delineating between setting short- and long-term goals. The ICDP online system used by HINAP provides an overview of how to develop “SMART” goals (Specific, Measurable, Achievable, Realistic and Timely; Fuhrmann et al., 2013). HINAP also specifies additional job search skills including interview skills, negotiating job offers and learning how to read job advertisements.\n\nIn sum, PSP and HINAP both offer a range of similar career development activities for biomedical doctoral and postdoctoral trainees that enable them to expand their career options, connect with role models, and begin developing a number of job search skills and set career planning goals. Each program is unique with respect to PSP’s emphasis on self-discovery using discussions and narrative development that aligns with more recent constructivist and life design approaches to career development. Alternatively, HINAP uses a more traditional, structured approach that relies on an online career information system that was designed specifically to explore biomedical science careers.\n\n\nCourse evaluations\n\nEach institution conducted independent evaluations of their respective courses. Both institutions used online surveys, and each conducted a nonrandomized repeated measures pretest-posttest design. This design is classified as a “pre-experimental” by Campbell et al. (1963), which means that while the results can be used to assess whether the courses show promise in supporting biomedical graduate student and postdoctoral fellow career development, the evidence should be considered correlational in nature and therefore one cannot draw conclusions regarding cause and effect. Each evaluation varied significantly and therefore the method and results are presented separately. Collective and noteworthy trends and their implications are addressed in the discussion, along with recommendations for future courses.\n\n\nStudy 1 – Preparing Science Professionals (PSP) course\n\nBased on the thematic analysis, the PSP career development course aims to support biomedical doctoral and postdoctoral trainees’ ability to engage in self-exploration of their skills, career exploration by examining both academic and non-academic career pathways, and the development of career management and planning skills. The purpose of this evaluation study was to assess whether participation was associated with increased awareness of their transferable skills and a wider range of career options, career management self-efficacy, and self-efficacy associated with pursuing academic and non-academic career opportunities.\n\nParticipants. A total of 32 respondents completed pretest and posttest evaluations (of 32 who were enrolled; see Underlying data – S1 & S2 (Layton et al., 2020)). Two respondents were missing data from a single timepoint due to missing either a pre- or post- survey. PSP course participants were informed of the study’s purpose and their rights as research participants using an emailed cover letter that also included a link to respond, and consent to participate was indicated by choosing to complete the survey. Participants were asked to select gender from a list of possible identities and 61% identified as female, 36% as male, and the remainder (<3%) identified as transgender or declined to identify a gender preference. Among the respondents, 44% were enrolled in a 17-week semester-long version of the course, whereas 53% were enrolled in a 7-week version of the course, and 3% of the respondents did not indicate which course was completed.\n\nParticipants included students enrolled in doctoral (83%) or Master’s (13%) programs with the remining being post-doctoral researchers (3%). The majority of course participants enrolled in the courses were from the Integrated Biomedical Sciences umbrella program (53%), followed by Biology (6%) and Nutritional Sciences (6%), and the remainder included representation equally spread across the following programs (3% each): Anthropology, Chemistry, Civil Engineering, Education, Integrated Plant/Soil Sciences, Nursing, Pharmaceutical Sciences, Physiology, Public Health, Social Work, and Toxicology/Cancer Biology. Most graduate students were in the 1st or 2nd years of their respective programs (M=1.65, SD=1.25; ranged from 1-7 years in program, including a 7+ option).\n\nEthics. Exempt status was sought and obtained through the Institutional Review Board of the respective institutions (University of Kentucky, IRB protocol #: 16-1034-X2B). All analyses were conducted on de-identified data sets to maintain confidentiality (see Underlying data: S1 & S2 (Layton et al., 2020)).\n\nMeasures. Measures were rated using a five-point scale from 1 (Strongly Disagree) to 5 (Strongly Agree), unless otherwise noted.\n\nConfidence to Pursue an Academic or Non-Academic Career. Two items were used to assess confidence for pursuing either an academic or nonacademic career. The first item asked, “To what extent [you are] confident [that you] understand the process, materials, and skills that are needed to transition into and excel in an academic career (i.e., faculty career path).” Using the same format, the second item asked about confidence to transition and excel “in a career outside academic or research (i.e., alternative or non-traditional career).” Each item was evaluated separately to assess whether course participation was associated with an increase in confidence toward pursuing each career pathway, respectively. Because the two single-item responses were considered separately, internal consistency estimates are not applicable.\n\nAwareness of Transferable Skills. Participants were asked to evaluate the extent to which they believe they had developed 15 transferable skills including: “discipline-specific knowledge”, “ability to gather and interpret information”, and “ability to analyze information.” The 15 items were summed with higher scores reflecting more awareness of transferable skills (Sinche et al., 2017; Sinche, 2016). Internal consistency using Cronbach’s Alpha was .72 at pretest and .83 at posttest.\n\nCareer Management Self-efficacy. A total of 10 items assessed confidence to engage in career management activities. Sample activities included “conduct job interviews,” “identify job openings,” and “effectively pursue a career path.” Items were summed with higher average scores indicating higher career management self-efficacy. Internal consistency using Cronbach’s Alpha was .74 at pretest and .92 at posttest.\n\nBiomedical Career Path Familiarity. Using a four-point scale ranging from 1 (Not at all familiar) to 5 (Very familiar), participants were asked to rate how familiar they were with 15 career paths (mirroring NIH BEST Consortium baseline survey; Lenzi et al., 2020). The items were summed with higher average ratings indicating more career path familiarity. Internal consistency using Cronbach’s Alpha was .72 at pretest and .83 at posttest.\n\nCareer Choice. Participants were asked to select one of the 15 career choices provided or to indicate “other” accompanied with a free-response text box. Sample career choices included “faculty academic research,” “teaching,” “policy,” and “academic administration.” This item was evaluated with respect to whether participation was associated with changes in the individual’s career choice selection pre- versus post-course participation, and whether there were shifts in the overall percentages in selected careers. To assess whether the course participation was associated with a change in one’s career, participants received a “0” if their post-course career choice matched with their pre-course career choice, and received a “1” if their post ratings reflected any change in career choice.\n\nCareer readiness. Paired-sample t-tests (Tabachnick & Fidell, 2007; all analyses conducted using IBM SPSS) were used to assess whether participation in a biomedical career development course was associated with Confidence to Pursue an Academic or Non Academic Career, Awareness of Transferable Skills, Career Management Self-efficacy, and Biomedical Career Path Familiarity, respectively (see Table 3). Following completion of the course, participants reported increased Confidence to Pursue an Academic (t[28] = 6.25, p < .001) and Non-Academic Career (t[28] = 4.34, p < .001), Awareness of Transferable Skills (t[28] = 3.41, p < .01), Career Management Self-efficacy (t[26] = 7.74, p < .001), and Biomedical Career Path Familiarity (t[24] = 7.60, p < .001).\n\nCareer readiness composite variable summaries pre- and post-course participation (means and standard deviations).\n\nCareer pathway. Prior to the course, trainees indicated an interest in the following careers (3 responses were missing), of those who responded: 31% indicated non-academic research (n=9); 24% indicated teaching faculty (n=7); 10% (n=3) each indicated teaching/outreach, industry administration, and consulting; 3% (n=1) each indicated policy or regulatory affairs; 7% (n=2) indicated other.\n\nPost course trainees continued to indicate an interest in the following careers (1 response was missing): 19% non-academic research (n=6); 16% industry administration (n=5); 13% teaching faculty (n=4); 10% (n=3) each indicated teaching/outreach, policy, and tenure track academic; 7% academic administration (n=2); 3% (n=1) each indicated non-faculty academic research-focused, consulting, entrepreneurship/startups, or medical science liaison, and other. Of these, the following were indicated post course, but not selected initially pre-course by any respondents: tenure track academic, academic administration, non-faculty academic research, entrepreneurship/startups, and medical science liaisons.\n\nCourse role in changing career pathways. A little over half of the respondents (54%) changed career preference over the duration of the course (46% remained the same; see Figure 1). The category of “other” was reduced in half from pre- to post-course (7% to 3%); however, given the small sample size this should be interpreted with caution.\n\nPSP participants were surveyed before (blue) and after (red) participating in the course about their career interest areas. NR designates no responses for that item.\n\nIn summary, results suggest that PSP participants’ career readiness increased on various measures of career efficacy, including pursuing an academic or non-academic career, awareness of transferable skills, career management self-efficacy, and biomedical career path familiarity. Further, trainees continued to be interested in several different types of career paths. Interestingly, about half of the participants changed their career preference with some indicating careers that were not selected at the beginning of the course, including tenure track faculty.\n\nIn Study 1, the PSP career development course sought to help biomedical trainees explore how the skills being developed could be applied across a wide variety of academic and non-academic career pathways as well as career management and planning skills. Examination of pre- and post-course results indicated that participation in the PSP course was associated with increased self-efficacy related to pursuing academic and nonacademic job opportunities, respectively, as well as increased familiarity with biomedical career pathways. Participants also reported more awareness of how the transferable skills they were developing in the graduate programs and more confidence in career management and planning. While the sample size (n = 32) is small, the ability to detect significant increases in posttest ratings is an indication that the career information and pedagogy effectively met the goals of the course. It is important to note the lack of a comparison group, which makes it impossible to draw cause and effect conclusions. However, the evaluation of the evidence supports the conclusion that course participation is associated with increasing career development. In order to draw conclusions about the impact or causal nature of the course, future evaluations should consider using a separate-samples pretest-posttest design whereby participants are randomly assigned to be observed before or after the course. More details on the applicability of this assessment strategy will be described in the General discussion section.\n\nThree points are especially noteworthy regarding career pathway trends and changes. First, a greater variety of career interest areas were endorsed after (12 total career paths) compared with before (8 total career paths) the course, suggesting that familiarity with a wider number of career options developed during the course. Second, if “other” is interpreted as “undecided,” then the decrease in this category from pre- to post-course may indicate the selection/identification of specific career interest that developed during the course. In this case, as no “undecided” option was available, this interpretation seems plausible. Third, and perhaps surprisingly, interest in faculty careers increased after familiarizing trainees with other career options.\n\n\nStudy 2 – Hope is Not a Plan (HINAP) course\n\nThe HINAP career development course centers around effective use of an existing online resource, an Individual Career Development Plan (MyIDP) to help biomedical trainees to examine how well their skills and interests match to 20 scientific career paths and facilitates users to set short and long-term goals related to pursuing identified career paths of interest. The course is typically 8 weeks long for a total of 12 hours (initial versions of the course ran 10 weeks for 15 hours but has since been modified). On average, course enrollees attended approximately 7 of the 8-10 sessions offered in a semester (M = 6.88, SD = 1.63).\n\nParticipants. A total of 148 respondents (of 359 who were enrolled; see Underlying data – S3 & S4 (Layton et al., 2020)) completed pre and post data over a five year period. The HINAP course participants were informed of their rights as research participants at the start of the survey, and consent was indicated by confirmation of intent to participate at the conclusion of an embedded one-page summary. The course is offered both as a required graduate course in some programs as well as an elective course open to both doctoral students and postdoctoral fellows. The current sample represents 10 course sections (mode = 18 responses per section, ranging from 5-35 responses each). A subset of respondents (n = 14) were missing demographic data responses, including department, gender, etc. Of those who responded, participants identified as female (63%), male (36%), or elected not to provide a response (1%). The average age of the sample was 29 years (M = 29.16, SD = 3.99; ranged from 23 to 47 years of age). About half identified as US Citizens (42%), with a large international contingent (55%; including Green Card holders, J1/F1 visas, H1B visas, etc.).\n\nSlightly over half of the enrolled course participants were graduate students (56%), with the remainder identifying as postdoctoral trainees (44%). The majority of participants (83%) identified as biomedical scientists from a variety of departments (specifically: 26% Biomedical Sciences umbrella program, 7% Neuroscience, 7% Biochemistry, 5% Microbiology and 18% other biomedical - e.g., Radiology, Neurology, Surgery, Pathology, Population Health, Medicine, Dental, and 20% Biology). The remaining 17% included other primarily STEM departments such as 10% Chemistry, with 7% of responses from a variety of other departments (e.g., Psychology, Mathematics, Population Health, Basic Sciences, Physics, Mechanical Engineering). Graduate students were typically in their 2nd or 3rd year of training (M = 2.90, SD = 0.97; range 1–6 years) and postdoctoral trainees were typically in their 1st or 2nd postdoctoral position (M = 1.33, SD = 0.57, ranged from 1–3 positions).\n\nEthics. As with Study 1, exempt status was sought and obtained through the appropriate Institutional Review Board (New York University, IRB protocol #: I13-00727). All analyses were conducted on de-identified data sets to maintain confidentiality (see Underlying data: S3 & S4 (Layton et al., 2020)).\n\nMeasures. Career Readiness. A composite variable was created to approximate Career Management Efficacy by summing three items, to which respondents could endorse or not endorse (yes or no). These items evaluated whether trainees knew where to look for jobs in their desired field, understood how candidates are evaluated for jobs in their chosen field, and whether they felt prepared to seek a position in their desired field (mean responses range from 0 to 3).\n\nCareer Professional Mentoring. Participants were asked to indicate who served as their own mentors (including PI, faculty, professional, and/or peer) pre- and post-course participation. The number of mentors cited by trainees was compiled into an arithmetic sum; in addition, changes in endorsement of each category were also examined. Career staff mentors were only included in the post measure, hence results are presented both with and without including that option when comparing pre- to post-mentorship measures.\n\nCareer Pathway. At the beginning and end of the course, participants were asked to select the career pathway most closely aligned with their current career goal from 10 specified options, plus “not sure,” or “other” with a free-response text box. Career options included tenure-track faculty; other academic, research or teaching; private industry, research or non-research; government/non-profit; science writing, publishing, and communications; law; consulting; and entrepreneur.\n\nReason for Changing Career Goal. Participants were asked whether they had changed their career goal as a result of participating in the course. If they responded affirmatively, participants were then asked to explain “why has your career goal changed.”\n\nThe HINAP course evaluation strategy included measures of career confidence, career and professional mentorship, career path familiarity, and included a rich commentary descriptive in nature, hence the results include qualitative themes that explore whether and how participation in the course was associated with helping biomedical trainees consider diverse career pathways.\n\nCareer readiness. Career Management Efficacy significantly improved from pre- to post-course on the composite variable (see Table 4 for individual item response trends), t(132) = 18.65, p<0.001), with trainees agreeing with fewer than one of three questions on average pre-course (M=0.64, SD=0.73) and agreeing with two out of the three questions on average post-course (M = 2.03, SD=0.86).\n\nCareer readiness composite variable questions, percentage of yes endorsements by item (increase or decrease noted).\n\nCareer professional mentorship. The overall number of mentors cited by trainees (including PI, faculty, professional, and/or peer) increased significantly from pre- to post-course (M=1.65-1.95, t(137) = 3.09, p=.002, Table 5). In addition, the pattern of mentorship increase was maintained and the effect was enhanced when program mentors (i.e., Science Training Enhancement Program mentors) were included as a mentor option (M=1.65-2.10, p<.001). Furthermore, the number of trainees who cited having no mentors decreased, with 15% of participants (n=20) citing having no mentors pre-course and only 5% (n=7) post-course. In addition, the number of people who endorsed having a contact (or mentor) in their career field of interest rose from pre- to post-course from 24% to 70% (although the wording was slightly different, so direct statistical comparison is not appropriate for this question). Although these results should be interpreted with caution, since it’s impossible to rule out confounding factors (e.g., mentor vs. contact; having identified one’s field of interest during the course; meeting staff or faculty instructors; etc.), the pattern of increasingly diverse mentorship is consistent with the results from identical pre- and post-survey, which show gains of similar magnitude across almost every category (higher percentages endorse having mentors of each type). Ratings of trainees’ own PI engagement remained constant (no significant change from pre- to post- course, M=3.48-3.43, t(81)=-.54, p=0.59, NS), suggesting that PI support and mentorship remained consistent throughout this career exploration process – even while the overall number of mentors that trainees reported having available to them increased concurrently.\n\nCareer and professional mentorship endorsements by category increased pre-to post-course participation, suggesting that more mentors were identified during course participation (increase or decrease noted).\n\nCareer choice. At the beginning of the course, participants were asked “Right now, what is your main career goal” (see Figure 2). Given that the HINAP course is focused on exploring nontraditional career options, it is not surprising that 36% of the participants indicated either “Not sure” or did not respond the question. Of the 95 who did identify a career pathway, 38% (n = 36) indicated Private Industry; 27% (n = 26) indicated Tenure-Track Faculty; 11% (n = 10) indicated Other Academic Research or Teaching; 9% (n = 9) indicated Consulting or Entrepreneurial; 8% (n = 8) indicated Science Communications; 6% (n = 6) indicated Government/Non-profit; and <1% selected Law (n = 1) or Medicine (n = 1).\n\nHINAP participants were surveyed before (blue) and after (red) participating in the course about their career interest areas.\n\nWhen asked the same question after the course was completed post-course participation (“Now, what is your main career goal”), only 18% (n = 27) responded “Not Sure” (n=26) or did not respond (n = 1). Of the 121 participants who did indicate a career pathway, 40% (n = 48) indicated Private Industry; 29% (n = 35) indicated Tenure-Track Faculty; 9% (n = 11) indicated Other Academic Research or Teaching; 9% (n = 11) indicated Government/Non-Profit; 6% indicated Consulting or Entrepreneurial; and 2% (n = 3) indicated Law (n = 2) or Medicine (n = 1).\n\nIn sum, the results indicate two trends. The first trend was that course participation was associated with a decrease in “unsure” or blank responses – from 36% at pretest to 18% at posttest. The two most popular pathways – industry and tenure-track faculty – remained the most popular. Specifically, an increase from 36 to 48 participants (2% increase) indicating private industry and an increase from 26 to 35 participants (2% increase) indicating tenure-track faculty. Of note, as observed in Study 1, following participation in the course a subset of participants moved into the Tenure Track Faculty category from other categories, indicating again that career exploration courses did not discourage participants from pursuing academic careers but enhanced interest in some cases.\n\nCourse role in changing career pathways. With respect to whether participation in the course was associated with changing participants career pathway goals, among the 148 respondents, 58% reported “No” or “Not Sure” and 42% indicated “Yes.”\n\nA total of 37 participants provided written responses about the course role in supporting their career exploration, choice, and decision-making (see de-identified responses in Underlying data: S3 & S4 (Layton et al., 2020)). For some participants, responses indicated that the course helped them clarify their career goals and intentions. Sample responses include:\n\nAlthough the official job title associated with my main career goal has not changed, how I intend to fill this role has changed, which influences the higher-education institutions I am interested in.\n\nAlthough I am still unsure, the course helped me identify some good career path matches and even learn about careers that I did not know science PhDs could pursue.\n\nDebating between non-profit/outreach and industry and realized would be happier doing research in biotech setting.\n\nDon't really have a desire to go into consulting after researching the work/life balance. I am leaning toward for-profit industry either on or off the bench, not sure what yet.\n\nI am definitively considering more seriously a career in research in a private company.\n\nI am not sure what my career goal is but the class made me consider my values and how they would impact my job choice.\n\nI didn't change my career goal really but I learned a lot about how to positively influence my chances at getting a job that I like.\n\nOther responses indicated that the course expanded their consideration of career pathways outside of academia.\n\nI got to know possibilities in the private industry, with better wages and faster dynamics.\n\nIt verified that I want to pursue a career that has less to do with the academic setting, a more with the regulatory aspects of small molecule regulation in food products, environment, etc...\n\nI was already becoming less interested in Industry research but this class helped me come to the conclusion that I will probably pursue patent law.\n\nSome respondents indicated that the course helped them understand how to evaluate their options and set goals for further examination and clarification.\n\nIt didn't affect my primary goal, but definitely provided new insights into the process of applying and interviewing for academic jobs. The class also encouraged me to keep an open-mind and to explore various other career paths I'd been considering.\n\nIt certainly helped me sit down and consider my options, my skill set and what aspects of my work I enjoy the most - and how that could become a full-time career! I've got a few options now that I am hoping to pursue.\n\nIt helped me figure out ways to get the information needed to really determine which of the paths I have been considering (tenure track faculty, industry, or senior scientist) is the best fit for me.\n\nThis course has actually made me to sit down and write down my short and long term goals that has helped me to focus in choosing my career.\n\nIn summary, HINAP participants’ career management self-efficacy and career and professional mentorship increased, with broadened career pathway selections including an increase in participants who were interested in tenure track research faculty roles. Furthermore, qualitative data indicated that the course supported participants’ career exploration through clarification of career goals; exploration of pathways outside of academia; and evaluation of career options with goal-setting to support career decision progress.\n\nThe HINAP course sought to expand biomedical trainees’ awareness of career pathways that lie outside of academia and to help them set goals for pursuing their career pathway goals. Career readiness increased across each of the three items assessing career management self-efficacy, resulting in a significant increase for the composite variable. Mentorship options were broadened for participants from pre- to post-course: total number of mentors significantly improved; the number of those identified as lacking mentors decreased; and individuals identified as career and professional mentors and career field contacts increased. Virtually all categories of mentorship showed increases (with the exception of PI, which did not change). This suggests that career course participation may encourage trainees to identify potential mentors, reach out to mentors in a variety of categories, and/or to recognize existing mentorship relationships. As with Study 1, a wide variety of career choices were selected; the choice of tenure track research faculty increased; and the choices of unsure and other decreased. While a large percentage of participants selected private industry and tenure-track career pathways both pre-and post-course, the open-ended responses indicated that the course helped participants examine a broader range of career options and helped identify goals for further exploration as they pursue their selected career pathways. Finally, qualitative responses indicated support for career goal-setting, exploration, and clarity were provided through course participation.\n\n\nGeneral Discussion and Recommendations for Future Course Implementation\n\nThe number of trainees seeking graduate study in the biomedical sciences continues to rise and the number of available tenure-track faculty positions continue to wane, with the net result of many graduates extending post-doctoral positions without a clear plan or opportunity for future job security. In response to this challenge, NIH and National Science Foundation have both encouraged graduate programs to introduce career development offerings that will enable trainees participating in doctoral and post-doctoral programs the opportunity to explore how their skills can transfer into a wider range of high paying career pathways outside of academia. This study evaluated the thematic content and course evaluations for two independently developed, complimentary academic courses career development courses for doctoral and postdoctoral trainees offered at the two distinct institutions.\n\nThe thematic analysis evaluated both courses in terms of how they facilitated the development of self-exploration skills, career exploration skills, and career planning and management skills. Self-exploration skills refer to activities that enable participants to become aware of their transferable skills, interests, and values before engaging in career exploration skill activities that help participants match these transferable skills to a range of occupations. Career planning and management refers to setting goals and developing plans for further exploration and for identifying and pursuing specific job opportunities. The results of the thematic analysis indicated that the PSP course emphasized a more narrative and constructivist strategy for helping participants engage in self-exploration using discussions. Alternatively, the HINAP course focused on effectively implementing tools from the online platform, relying on assessment tools for helping participants identify their skills which then matched their responses to a range of career opportunities. To facilitate deeper career exploration, the PSP course used role models who described their career journey as a way to introduce alternative career pathways. The HINAP model focused on establishing SMART goals as part of a career management and planning effort.\n\nThe two courses varied considerably in how they conducted their course evaluation. The PSP course relied on the use of quantitative measures that assessed constructs such as self-efficacy, career management, and familiarity of careers. While career management self-efficacy was also measured for the HINAP course, course assessments focused on identifying how course participation impacted their career choice and gathered qualitative responses associated with participants career choice and decision-making process.\n\nThe PSP course pretest-posttest design found that course participation was associated with participants reporting more confidence with respect to pursuing both academic and nonacademic career pathways. Participants reported more familiarity of biomedical pathways, and participation was associated with increased self-efficacy related to pursuing both academic and nonacademic job opportunities and career management and planning.\n\nThe HINAP course also showed an increase in participants’ career management self-efficacy, but the primary course focus was on identifying whether participants expanded their career pathway choices, and to better understand whether and how the course facilitated their career exploration and goal setting. The two largest career pathways were private industry followed by tenure-track faculty. Results indicated that participation in the course was associated with reducing in half the percentage of participants who were uncertain about their future career pathways with 18% of the course participants indicating being unsure of their career pathways following the course compared to 36% being unsure when the course began. Qualitative responses offered insights into the number of ways in which the course supported participants in being able to clarify their career goals, deepen their understanding of their selected career pathways, and provide clear goals for further exploration and the job search process.\n\nUniquely, the HINAP course measured pre- and post-course participants’ access to career and professional mentorship, which increased over the course of their participation. Principal Investigator (PI) engagement in mentorship showed no change pre- to post- course, which might be interpreted either as continued PI involvement in mentoring their trainees, irrespective of trainee participation in the course. Given the variety of demands on PIs time, it may be seen as a benefit to have trainees increase their mentorship network, thereby taking some pressure off of PIs to be a one-size-fits-all mentor for every career path. While undoubtedly PIs should play a primary advising role for every mentee in their lab, due to practical time limitations and lack of PIs experience outside academia, it may be unrealistic to expect PIs to serve all these advising roles simultaneously.\n\nThe impact of peer mentorship is another topic that has yet to be systematically examined in a career-focused setting. While trainees asking peers about job search experiences and sharing information as peer-mentors occurs anecdotally, the impact has yet to be quantified formally (Kram & Isabella, 1985). Our data suggest that the vast majority of trainees do indeed turn to peers for support and mentorship in the career planning paradigm (73-83% of trainees). Another important implication is that peer-to-peer training opportunities could be more formally encouraged, including leadership roles and career development opportunities for senior trainees; this could enhance the resources available to trainees as they are navigating the career development planning process (Dorman et al., 2018).\n\nIn addition, while the data is inconclusive, administrative career-focused staff (e.g., career and professional development staff) may have become a newly utilized resource – and almost certainly added to the potential mentor-pool from which trainees may choose to draw. The development of additional resources to support doctoral trainee career transitions (e.g., NIH BEST programs; Lara et al., 2020; Lenzi et al., 2020; NIH, 2012) is of growing national interest (e.g., [Blank et al., 2017; Benderly, 2015; FOBGAPT, 2017; Stayart et al., 2018); one solution to workforce preparation in graduate education includes the use of staff trained to provide guidance and resources leading up to and during this transition to the workforce. Furthermore, the focus on career transitions and career outcomes is especially salient currently due to the federal focus that has recently been introduced to encourage development of more such programs (e.g., federal training grants such as NIH/NIGMS T-32 including career development training requirements and career outcome reporting; Gammie et al., 2017); hence, it seems likely that the importance of shepherding trainees through career transitions will only continue to gain importance.\n\nIncreasing the interface between industry partners and professionals outside of academia is crucial. Across two measures of STEM PhD professional mentorship and contact endorsements, a positive shift was identified. Programs like NIH BEST and other sustained programs that develop on-going, long-term partnerships across professional fields are crucial to provide readily available, existing, robust networks of professionals that trainees can tap into. Career courses are one way to enhance these types of connections. While PI introductions to industry professionals can also be a direct route to connect with STEM PhDs in other fields, the opportunity for these may be limited to the personal connections of the individual trainee (or connections of the PI), despite the best intentions of both. Hence the recommendation to enhance the connections available to graduate and postdoctoral trainees via programmatic partnerships as a whole, which will facilitate departmental and institutional access to professionals across a variety of careers, to expose and connect trainees with. Career services and alumni offices may also be of help in these cases, as can access to alumni directories through each institution, and/or electronic networking connections (e.g., LinkedIn alumni search tool).\n\n\nRecommendations\n\nBoth courses offer distinct, yet complimentary, versions of career development courses designed to help biomedical doctoral and postdoctoral trainees identify career pathways. While the PSP course emphasized self-exploration discussions and exposure to role models who used their doctoral degree to pursue a wide range of academic opportunities, future course design may want to consider incorporating HINAP course efforts to facilitating deeper career exploration. Career exploration could be enhanced through discussions or formal assessment of the RIASEC personality type (Holland, 1958; Holland, 1959) and by providing trainees access to an online career information system such as the individualized career development plan (ICDP) or O*NET. By using online systems, trainees will have access to more descriptive details and labor market information that may not be available comprehensively from role models, as well as offer additional opportunity to align their interests and RIASEC type to a wide range of career opportunities. During the final third of the course, PSP could also incorporate HINAP’s SMART goal exercises as a more explicit career management and planning effort. SMART goals should include both future work and balancing home and life related concerns.\n\nFor HINAP, more emphasis could be placed on engaging in self-exploration prior to making career choices and setting career (SMART) goals. Incorporating PSP’s strategy for using role models and having conversations about past, present, and future roles and life goals would enable a rich self-exploration foundation that is likely to facilitate career exploration and goal setting.\n\nWith respect to evaluation, both courses could benefit by incorporating a more rigorous, quasi-experimental, evaluation design. The separate-samples, pretest-posttest design described by Campbell & colleagues (1963) has been used in studies focused on interventions (e.g., increasing exercise, Amaya & Petosa, 2012; managing stress, Payrau et al., 2017; among others). With respect to career development, Solberg and colleagues used a separate-samples pretest-posttest strategy to verify the impact of a social emotional learning intervention (Solberg et al., 1998) and the effects of engaging in individualized learning plans (ILPs) on participants’ career search self-efficacy and career readiness (Solberg & Gresham, 2011). Rather than randomly assigning participants to a treatment or control group, separate-samples, pretest-posttest design randomly assign whether participants complete a pretest or posttest. By designing a mixed methods evaluation strategy that incorporates both PSP’s quantitative measures and HINAP’s qualitative measures, it is possible to randomly assign participants to complete the quantitative assessment at pretest followed by the qualitative assessment at posttest or complete the qualitative assessment at pretest and quantitative assessment at posttest. Rather than conducting the pretest-posttest solely across a whole semester or summer term, future course evaluations could discretely measure each of three modules – self-exploration skills, career exploration, and career planning and management.\n\n\nConclusions\n\nIn summary, these studies offer two complimentary course design strategies for improving career readiness among doctoral and post-doctoral trainees. A thematic analysis of the courses indicated two complimentary but different approaches to career development. Using ILPs as a guiding framework, the first institution (PSP course) incorporated a number of life design elements by creating opportunities for discourse among course participants and professional role models. The second institution (HINAP) focused on examining interests and setting goals using the online career information myIDP. Using a quantitative pretest-posttest evaluation, we found that participants in the PSP course reported more awareness of how their PhD skills transfer into a wide range of career opportunities both in and outside of academia and they reported higher career management skills. The HINAP course employed a more qualitatively rich approach to evaluating career readiness, revealing that participants explored a broader range of career options and established career planning goals.\n\nA perhaps surprising finding from examination of both courses was that in both cases, a subset of participants became interested in pursuing traditional tenure-track academic positions. A concern expressed by some faculty has been that broad career exploratory courses may discourage students from pursuing academic positions. Here, to our knowledge for the first time, we present data that the opposite is true – exposing trainees to the large breadth of career options available to them can actually enhance interest in academic careers for a subset of students.\n\nAs was supported by course participation in at least one of the courses, career exploration and professional development networks can be supported by building relationships with professionals in the field. Hence, building career mentor networks could also be a goal of future career courses in addition to the focus of exposing trainees to a myriad of career pathways and materials to explore them.\n\nFurthermore, broad career exploration opportunities during research training should enable trainees to better identify and assess potential careers that are the best fits for their individual interests, preferences, and skills. In sum, offering graduate and postdoctoral students access to courses that enable them to expand awareness of careers that align with their advanced skill sets was beneficial in either supporting their ability to select new career options or to increase confidence in pursuing preexisting career choices.\n\n\nData availability\n\nDe-identified data is available. As per IRB limitations on data sharing to protect the identities of participants, all personally identifying information including demographic data has been removed, though it is reported in aggregate in the manuscript.\n\nOpen Science Framework: Career planning courses increase career readiness of graduate and postdoctoral trainees - Extended Data S1-6, https://doi.org/10.17605/OSF.IO/9WRBY (Layton et al., 2020).\n\nInformation about each variable is embedded within the data files (SPSS version), removing the need for a data key; however, open source format versions (CSV) are also included for increased accessibility to data. Pre- and post-course survey questions are also embedded in the SPSS data files. This project contains the following underlying data:\n\nExtended Data File – S1. PSP De-identified Data (SPSS)\n\nExtended Data File – S2. PSP De-identified Data (CSV)\n\nExtended Data File – S3. HINAP De-identified Data (SPSS)\n\nExtended Data File – S4. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLayton RL, Jahangir AE, Solberg VSH, et al.: Career planning courses increase career readiness of graduate and postdoctoral trainees - Extended Data S1-6. 2020. http://www.doi.org/10.17605/OSF.IO/9WRBY\n\nLenzi RN, Korn SJ, Wallace M, et al.: The NIH “BEST” programs: Institutional programs, the program evaluation, and early data. FASEB J. 2020; 34(3): 3570–3582. Publisher Full Text\n\nMathur A: Meeting the Moment. Inside Higher Ed: Career Advice. 2020. Reference Source\n\nMathur A, Chow CS, Feig AL, et al.: Exposure to multiple career pathways by biomedical doctoral students at a public research university. PLoS One. 2018; 13(6): e0199720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcDowell GS, Gunsalus KTW, MacKellar DC, et al.: Shaping the Future of Research: a perspective from junior scientists [version 2; peer review: 2 approved]. F1000Res. 2014; 3: 291. 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J Vocat Behav. 2016; 97: 84–89. Publisher Full Text\n\nSavickas M, Nota L, Rossier J, et al.: Life designing: A paradigm for career construction in the 21st century. J Vocat Behav. 2009; 75(3): 239–250. Publisher Full Text\n\nSchwab K: The Global Competitiveness Report 2019. The World Economic Forum Insight Report. 2019. Reference Source\n\nSchnoes AM, Caliendo A, Morand J, et al.: Internship Experiences Contribute to Confident Career Decision Making for Doctoral Students in the Life Sciences. Cbe-life Sci Educ. 2018; 17(1). Publisher Full Text\n\nSchultheiss D: Qualitative Relational Career Assessment: A Constructivist Paradigm. J Career Assessment. 2005; 13(4): 381–394. Publisher Full Text\n\nSinche M: Next Gen PhD: A Guide to Career Paths in Science. Cambridge, MA: Harvard University Press. 2016. Reference Source\n\nSinche M, Layton RL, Brandt PD, et al.: An evidence-based evaluation of transferrable skills and job satisfaction for science PhDs. PLoS One. 2017; 12(9): e0185023. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolberg VSH: Making School Relevant with Individualized Learning Plans: Helping Students Create Their Own Career and Life Goals. Cambridge, MA: Harvard Education Press. 2019. Reference Source\n\nSolberg VSH, Ali SR (Eds.): The handbook of career and workforce development: Research, practice, and policy. Taylor & Francis. 2017. Publisher Full Text\n\nSolberg VS, Gresham SL: Pre-Post Evaluation of the ECG Portal. Report prepared for the Singapore Ministry of Education. Madison, WI: Center on Education and Work. 2011.\n\nSolberg VS, Gusavac N, Hamann T, et al.: The Adaptive Success Identity Plan (ASIP): A career intervention for college students. The Career Development Quarterly. 1998; 47(1): 48–95. Publisher Full Text\n\nSolberg V, Martin J, Larson M, et al.: Promoting Quality Individualized Learning Plans Throughout the Lifespan: A Revised and Updated ILP How To Guide 2.0. (Number #OD-23804-12-7504-11). Washington DC: Washington DC: Institute for Educational Leadership. 2018. Reference Source\n\nSolberg VS, Wills J, Redmond K, et al.: Use of individualized learning plans as a promising practice for driving college and career readiness efforts: Findings and recommendations from a multi-method, multistudy effort. Washington, DC: National Collaborative on Workforce and Disability for Youth, Institute for Educational Leadership. 2014. Reference Source\n\nStayart B, Brown H, Layton P, et al.: Applying Inter-rater Reliability to Improve Consistency in Classifying PhD Career Outcomes. [Preprint]. bioRxiv. 2018 [cited 2018 Nov 12]. Publisher Full Text\n\nStephan PE: How economics shapes science. Cambridge, MA: Harvard University Press. 2012. Reference Source\n\nTabachnick B, Fidell L: Logistic Regression. Boston, Massachusetts: Pearson. 2007.\n\nTornow JS, Kurose J, Lewis WJ, et al.: Dear Colleague Letter: Non-Academic Research Internships for Graduate Students (INTERN) Supplemental Funding Opportunity. Alexandria, VA: National Science Foundation. 2018. Reference Source\n\nVanderford N: Plot your course. Nature. 2017; 546: 443. Publisher Full Text\n\nVanderford NL, Evans TM, Weiss LT, et al.: A cross-sectional study of the use and effectiveness of the Individual Development Plan among doctoral students [version 2; peer review: 2 approved, 1 approved with reservations]. F1000Res. 2018a; 7: 722. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVanderford NL, Evans TM, Weiss LT, et al.: Use and effectiveness of the Individual Development Plan among postdoctoral researchers: findings from a cross-sectional study [version 2; peer review: 3 approved, 2 approved with reservations]. F1000Res. 2018b; 7: 1132. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "72974", "date": "29 Oct 2020", "name": "Gary S. McDowell", "expertise": [ "Reviewer Expertise Academic research workforce development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, the authors describe the assessment of career readiness development within the context of academic career courses at two institutions, particularly directed at graduate students and postdoctoral researchers, specifically designed to increase: career awareness; interest; and career-related confidence.\nThe studies are well-designed, and the authors describe the methods and results clearly and concisely. The authors give good descriptions of the limitations of the studies and what their results do or do not indicate, and the authors also provide suggestions for those working in this field on what to consider in future studies.\n\nI thought the paper was well-written and overall is suitable for publication, and the article is very useful in providing supporting evidence for the benefits of implementing structured career development efforts during PhD training and postdoctoral professional development. Here I have laid out some minor points and questions that arose during my reading of the paper, which I would be grateful if the authors could consider addressing.\nGeneral Comments: I very much appreciated the description of the Individualized Learning Plan (ILP) in the Career Course Thematic Analysis. Would it be possible for the authors to perhaps expand this discussion to compare and contrast the ILP with the Individual Development Plan (IDP), something that the community may be much more familiar with (and especially as IDPs are introduced later in the paper)? In that way it may be possible to demonstrate whether one builds on the other (given my understanding of the use of ILPs and IDPs in industry, this may be the case). If it’s not possible/considered appropriate to compare and contrast them, articulating why that is the case would be beneficial also. My impression is that a key strength of this paper is illustrating the utility the approaches seem to provide to develop career readiness in graduate students and postdoctoral researchers and a discussion of the utility of both of these tools would be of great interest to the article's likely readership.\nStudy 1 states: “Third, and perhaps surprisingly, interest in faculty careers increased after familiarizing trainees with other career options.” - is it possible for the authors to determine that it is familiarity with other career options that is the cause of this? Or does it perhaps suggest that participants grew more confident due to an increasing familiarity with the academic career paths? Is it possible that exposure to well-structured and evidence-based training on the academic career path is the cause of this, as opposed to perhaps largely anecdotal, subjective and opaque information that participants may have received before? I would be interested for reflections on this point, and indeed some of the participant responses provided for Study 2 could suggest that such may have been occurring there. The discussion on peer mentorship (where, perhaps, many participants have been getting career information from peers/word of mouth, which may not necessarily be correct) may also point to this. I believe this phenomenon may have been described elsewhere (or perhaps I have simply heard it articulated by career development professionals at universities, but it has not been written into the literature). The authors may be correct that in learning about other career paths, participants may have become more certain about the academic path: it could be a simple case of knowing what you don't want helps to clarify what you really do want. But, I am reminded of (Van Noorden, 20181) and the phenomenon of poor communication between PIs and trainees that affects a multitude of issues around training and career development. It may simply be that the approaches detailed in this paper increase academic career interest by providing clear information on what steps to take and how to think about it, in a structured way. I'm particularly interested, given the gender makeup of participants (in both, remarkably similar, with just over 60% being women), that this could even be pointing to gender biases in mentoring and the provision of guidance in professional career development. In short, any further reflections the authors could share on this would be appreciated, as I think they could have some answers to these points in their data.\nStudy 1 has very few postdoctoral researchers participating, compared to Study 2. Did the authors notice any differences viewing the data from that lens? I'm particularly interested in how the authors could relate this to some of Michael Roach's and Henry Sauermann's work: (Roach and Sauermann, 20172) in terms of graduate students and (Sauermann and Roach, 20163) in terms of postdoctoral researchers. It would be helpful to see whether these differing career stages have differing reactions in terms of the metrics described in this article.\nIt may be that there isn’t enough data to draw any meaningful conclusions, but were the authors able to see any differences based on gender, career stage, immigration status etc. where this data was collected? This does not seem to be referenced in the paper, so if it is not I would greatly appreciate some clarifying thoughts on whether anything was seen or not, particularly suggestions for how this could be addressed by researchers interested in these demographics in future studies.\nMinor Comments:\nThere is a possible typographical error: “To date, the design and implementation of career development programs and services in biomedical programs is only recently beginning to become broadly accepted beginning”.\n\nThe authors state that “This framework is a variation from the traditional themes of “career awareness,” “career exploration,” and “career preparation/career immersion” (e.g., Colorado, Massachusetts)” - I am unsure what (e.g. Colorado, Massachusetts) it is referring to?\n\nIn Table 1: The box with the heading “Career management and planning skills” also has “Goal setting” and “Job search” written in it, and I was wondering if it was a copy/paste error into that cell - based on the content of this row of the table, perhaps that should be in the cell immediately to the right?\n\nIn Study 1, under career pathways, is it possible to present the results simply in terms of n, rather than a percentage? The numbers are quite small, and I just didn't think having them described as percentages was useful, and also made the results a little harder to read.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7660", "date": "03 Feb 2022", "name": "Rebekah Layton", "role": "Author Response", "response": "Reviewer 1 - Gary S. McDowell Lightoller LLC, Chicago, IL, USA R1.1. In this study, the authors describe the assessment of career readiness development within the context of academic career courses at two institutions, particularly directed at graduate students and postdoctoral researchers, specifically designed to increase: career awareness; interest; and career-related confidence. The studies are well-designed, and the authors describe the methods and results clearly and concisely. The authors give good descriptions of the limitations of the studies and what their results do or do not indicate, and the authors also provide suggestions for those working in this field on what to consider in future studies. I thought the paper was well-written and overall is suitable for publication, and the article is very useful in providing supporting evidence for the benefits of implementing structured career development efforts during PhD training and postdoctoral professional development. Here I have laid out some minor points and questions that arose during my reading of the paper, which I would be grateful if the authors could consider addressing. AUTHOR RESPONSE 1.1: Thank you, we are glad you found the research and findings valuable and of interest to the field of graduate career development. We similarly hope that this proves useful for faculty, staff, and other career development practitioners who want to implement career courses at their own institutions. R1.2. General Comments: I very much appreciated the description of the Individualized Learning Plan (ILP) in the Career Course Thematic Analysis. Would it be possible for the authors to perhaps expand this discussion to compare and contrast the ILP with the Individual Development Plan (IDP), something that the community may be much more familiar with (and especially as IDPs are introduced later in the paper)? In that way it may be possible to demonstrate whether one builds on the other (given my understanding of the use of ILPs and IDPs in industry, this may be the case). If not possible/considered appropriate to compare and contrast them, articulating why that is the case would be beneficial also. My impression is that a key strength of this paper is illustrating the utility the approaches seem to provide to develop career readiness in graduate students and postdoctoral researchers and a discussion of the utility of both of these tools would be of great interest to the article's likely readership. AUTHOR RESPONSE 1.2: Thank you for noting the confusion between ILPs and IDPs, the following clarifying sentence has been added earlier in the manuscript, along with additional references noted and included in the revised manuscript: “ILPs can be used across the lifespan (Solberg et al, 2018), and in higher education a variant commonly referred to as an Individual Development Plan (IDP) has been developed (can be used across training levels; Tsai, Vanderford, & Muindi, 2018; how to use with PIs and research trainees, Vincent et al, 2015).” In addition, we thank the reviewer for noting additional articles of interest below that have now been added: Tsai, J.W., Vanderford, N.L. and Muindi, F., 2018. Optimizing the utility of the individual development plan for trainees in the biosciences. Nature biotechnology, 36(6), pp.552-553. Vincent, B.J., Scholes, C., Staller, M.V., Wunderlich, Z., Estrada, J., Park, J., Bragdon, M.D., Rivera, F.L., Biette, K.M. and DePace, A.H., 2015. Yearly planning meetings: individualized development plans aren't just more paperwork. Molecular cell, 58(5), pp.718-721. Furthermore, we agree that Vanderford et al 2018 a&b and Hobin et al 2012 are relevant; they have already been included in text as well as in the references. Optional additional refs: Vanderford, N. L., Evans, T. M., Weiss, L. T., Bira, L., & Beltran-Gastelum, J. (2018). Use and effectiveness of the Individual Development Plan among postdoctoral researchers: findings from a cross-sectional study. F1000Research, 7. (a) Vanderford NL, Evans TM, Weiss LT et al. Use and effectiveness of the Individual Development Plan among postdoctoral researchers: findings from a cross-sectional study [version 2; peer review: 3 approved, 2 approved with reservations]. F1000Research 2018, 7:1132 (10.12688/f1000research.15610.2) (a) Vanderford NL, Evans TM, Weiss LT, et al. : A cross-sectional study of the use and effectiveness of the Individual Development Plan among doctoral students [version 2; referees: 2 approved, 1 approved with reservations]. F1000Res. 2018;7:722. 10.12688/f1000research.15154.2 (b) Hobin JA, Fuhrmann CN, Lindstaedt B, et al. : You Need a Game Plan. Science. 2012. 10.1126/science.caredit.a1200100 Vincent, B.J., Scholes, C., Staller, M.V., Wunderlich, Z., Estrada, J., Park, J., Bragdon, M.D., Rivera, F.L., Biette, K.M. and DePace, A.H., 2015. Yearly planning meetings: individualized development plans aren't just more paperwork. Molecular cell, 58(5), pp.718-721. R1.3: Study 1 states: \"\"Third, and perhaps surprisingly, interest in faculty careers increased after familiarizing trainees with other career options\".\" - is it possible for the authors to determine that it is familiarity with other career options that is the cause of this? Or does it perhaps suggest that participants grew more confident due to an increasing familiarity with the academic career paths? Is it possible that exposure to well-structured and evidence-based training on the academic career path is the cause of this, as opposed to perhaps largely anecdotal, subjective and opaque information that participants may have received before? I would be interested for reflections on this point, and indeed some of the participant responses provided for Study 2 could suggest that such may have been occurring there. R1.4: The discussion on peer mentorship (where, perhaps, many participants have been getting career information from peers/word of mouth, which may not necessarily be correct) may also point to this. I believe this phenomenon may have been described elsewhere (or perhaps I have simply heard it articulated by career development professionals at universities, but it has not been written into the literature). AUTHOR RESPONSE 1.3-1.4: Thank you, this is an excellent point; in fact, we cannot determine causality based on our data. As the reviewer suggests, another explanation may be due in part to learning about other career paths, and in part due to increased familiarity with academic career paths. Indeed, participant responses that could reflect increased familiarity with academic pathway included examples such as, \"although the official job title associated with my main career goal has not changed, how I intend to fill this role has changed, which influences the higher-education institutions I am interested in.\" Similarly, comments that could reflect an increase understanding of the application process include, \"it didn't affect my primary goal, but definitely provided new insights into the process of applying and interviewing for academic jobs. The class also encouraged me to keep an open-mind and to explore various other career paths I'd been considering.\" While we cannot ascertain definitively based on the evidence herein, we have added the following sentence in the manuscript to address this point further: “This demonstrated that familiarizing trainees with accurate information about different career paths and the steps to pursue these was valuable, regardless of the career track chosen. While we cannot ascertain career choice causality based on the evidence collected for this study, the data suggested that focusing on the skills shared in multiple paths, including internal and external to academia, didn't incentivize choosing one or the other. Instead, the exposure to information during the coursework provided the tools to use the new knowledge to reevaluate the path that best fits trainees’ skills and values.” We have expanded on career mentoring expectations and potential mismatches between PI and trainee expectations, noting the Van Noorden (2018) survey results, adding the following to the manuscript: “While our data does not indicate any mentoring deficit from this perspective, the expanded network post-course does imply a perhaps unfulfilled need. Consistent with previous survey data that PIs believe they are available to discuss experiments and trainee careers (90%) whereas nearly a third of trainees disagreed with the same statement (Van Noordan, 2018), this may indicate a perceived gap in expectations or needs of trainees for career mentorship provided, which an extended mentoring network could help address.” While we focus on the overall growth of the mentoring network for course participants, it is also noteworthy that professionals in the field and career development staff grew to the greatest extent; this may reflect the greater access these two groups have to career advising information, especially for careers outside of academia (versus faculty members who may have extensive academic career advice, or peers who may not have direct access to such experience yet). Van Noorden (2018). Leadership Problems in the Lab. Nature, 557, 294-296. doi: https://doi.org/10.1038/d41586-018-05143-8. R1.5: The authors may be correct that in learning about other career paths, participants may have become more certain about the academic path: it could be a simple case of knowing what you don't want helps to clarify what you really do want. But, I am reminded of (Van Noorden, 2018) and the phenomenon of poor communication between PIs and trainees that affects a multitude of issues around training and career development. It may simply be that the approaches detailed in this paper increase academic career interest by providing clear information on what steps to take and how to think about it, in a structured way. AUTHOR RESPONSE 1.5: Finally, the data does not distinguish between desirability of career path versus a better understanding of the career path. Hence, we can’t say for certain if it was exploration or planning that impacted changing in career pathways. Future research could better ascertain which aspects of career development differentially impact career choice itself; confidence in any given career choice; and effective career preparation. Nonetheless, interestingly, our data is in line with Sauerman and Roach (2017), who found that 5% of early to late stage PhD students moved from not interested to interested in a faculty career path, though it is unclear if experience, changing values/priorities, or career development/training is driving this effect (though the authors argue it is not likely attributable to differing career outcome expectations as they frame the question as independent of job market availability – though this is hard to rule out entirely as the authors note that career path changes could be chosen due to job availability in the first place). Our study suggests that at least in our sample, some course participants similarly transitioned from not interested in a faculty career path to interested in a faculty career path – importantly, this is after exposure to career exploration and planning during the course as compared with before the course. While we can’t conclude which aspect of the course content and training experience influenced their decision, it is still noteworthy that career exploration and planning activities are at the very least not turning people away from faculty careers – in fact, the opposite is true at least for some who were inspired to indicate interest in an academic faculty career after but not before participating in the career courses. This trend is of course based on a small n and hence must be interpreted cautiously, but the fact that it shows up as a trend across two independent samples is a sign that this is not likely an anomaly. To compare these results, the following reference has been added: Roach M, Sauermann H. The declining interest in an academic career. PloS one. 2017 Sep 18;12(9):e0184130. R1.6: I'm particularly interested, given the gender makeup of participants (in both, remarkably similar, with just over 60% being women), that this could even be pointing to gender biases in mentoring and the provision of guidance in professional career development. In short, any further reflections the authors could share on this would be appreciated, as I think they could have some answers to these points in their data. AUTHOR RESPONSE 1.6. We agree that the question of how gender in career development intersects with mentoring would be an interesting research question to explore. Unfortunately, we did not collect data on the quality or amount of mentorship the trainees received (rather simply the number of professional mentors endorsed pre- and post-course). This would be an excellent area for future research. Furthermore, to the reviewer’s point that our sample consists of proportionally more of women versus men, this is consistent with anecdotal data shared across the NIH BEST consortium (see Lenzi et al, 2020 for methods, entrance data, and program summary), for which gender distribution of voluntary participation was generally more heavily weighted toward women – and for our data also reflects a similar distribution. This would certainly be a direction for future research. While Lenzi and colleagues (2020) noted that, \"a remarkable majority of those interviewed revealed that they were reluctant to discuss their career plans with their mentors.\" Unfortunately, this observation was not separated by gender and should be addressed in future studies. Accordingly, we have added the following to the manuscript to indicate a call for such studies in future directions: “Our sample included a greater number of women participants than men, which has anecdotally been the case for NIH BEST programming (e.g., Lenzi et al, 2020). The question of how gender in career development intersects with mentoring would be an interesting research question to explore. The current data did not include ratings of the quality or amount of mentorship the trainees received (rather simply the number of professional mentors endorsed pre- and post-course). However, the impact of gender on professional development participation as well as differences in mentored career development experiences by gender, would be excellent area for future research.” R1.7: Study 1 has very few postdoctoral researchers participating, compared to Study 2. Did the authors notice any differences viewing the data from that lens? I'm particularly interested in how the authors could relate this to some of Michael Roach's and Henry Sauermann's work: (Roach and Sauermann, 2017) in terms of graduate students and (Sauermann and Roach, 2016) in terms of postdoctoral researchers. It would be helpful to see whether these differing career stages have differing reactions in terms of the metrics described in this article. AUTHOR RESPONSE 1.7. We agree with the reviewer that further elaborating on this point would be helpful—however, data collected regarding postdocs was insufficient. Specifically, Study 1 sample size for postdocs was too small to elucidate meaningful differences, and Study 2 had a primarily qualitative focus and wasn't designed to evaluate differences between graduate students and postdocs, hence this is a limitation of the current study and future work should address this important question. For this reason, we chose not to elaborate on this topic, but we added the following sentence in the general discussion: “In the present study, the number of participating postdoctoral trainees who completed quantitative measures was insufficient to conduct a robust analysis to determine if their experiences differed from graduate students. Postdoctoral fellows may lack of economic support to cover the cost associated with these courses and/or visa restrictions may affect participation in for-credit coursework. Hence, this is a limitation of the current study (for more on postdoctoral career outcomes, see Xu et al, 2018 and Silva et al, 2016; career interests may also differ between trainees with and without postdoctoral plans, Sauermann & Roach, 2016). Future work should address the critical question of how beneficial formal coursework is to postdoctoral trainees in contributing to their career development and exploration. Furthermore, longitudinal studies looking at the differential impact of career course participation across stages of training could better provide granularity of understanding of changing professional development effects.” We have, nonetheless, commented on our data in relation to the two sources mentioned by the reviewer (see also AR1.3-1.5 above in regards to Sauermann & Roach, 2017). Please note the additional reference added and described above: Sauermann H, Roach M. Why pursue the postdoc path?. Science. 2016 May 6;352(6286):663-4. R1.8: It may be that there isn't enough data to draw any meaningful conclusions, but were the authors able to see any differences based on gender, career stage, immigration status etc. where this data was collected? This does not seem to be referenced in the paper, so if it is not I would greatly appreciate some clarifying thoughts on whether anything was seen or not, particularly suggestions for how this could be addressed by researchers interested in these demographics in future studies. AUTHOR RESPONSE 1.8. Similar to our response above (see AR1.7), we appreciate the reviewer's insightful suggestion and agree that it would be useful to explore data and see If any differences are observed based on gender, career stage, Immigration status, but such an analysis is beyond the analyses possible for this study. We have added a call for such investigations in limitations and future directions, adding the following: “Future directions for research could include identifying differing trends or effectiveness of career course participation by gender, race/ethnicity, career stage, immigration status, and other variables best tested with a large sample size and high course enrollment. Future studies especially at institutions that require career development coursework of enrolled PhDs (e.g., as introduced by some NIH BEST Awardee institutions) would allow for robust analyses across multiple identity groups, while also reducing any potential self-selection bias of participants, which the current study cannot control for as these courses were offered as electives…. Furthermore, longitudinal studies looking at the differential impact of career course participation across stages of training could better provide granularity of understanding of changing professional development effects across graduate and postdoctoral career stages. R1: Minor Comments: R1 MC1: There is a possible typographical error: \"To date, the design and implementation of career development programs and services in biomedical programs is only recently beginning to become broadly accepted beginning.\" AUTHOR RESPONSE 1.MC1. Thank you for noticing this typographical error, it has now been corrected. R1 MC2: The authors state that \"\"This framework is a variation from the traditional themes of \"career awareness,\" \"career exploration,\" and \"career preparation/career immersion\" (e.g., Colorado, Massachusetts)\" - I am unsure what (e.g. Colorado, Massachusetts) it is referring to? AUTHOR RESPONSE MC2.The state-level distinction was not relevant for the purpose of this study; therefore, we have deleted the reference to it. R1 MC3: In Table 1: The box with the heading \"Career management and planning skills\" also has \"Goal setting\" and \"Job search\" written in it, and I was wondering if it was a copy/paste error into that cell - based on the content of this row of the table, perhaps that should be in the cell immediately to the right? AUTHOR RESPONSE MC3. This observation is correct; we have updated it accordingly. R1 MC4: In Study 1, under career pathways, is it possible to present the results simply in terms of n, rather than a percentage? The numbers are quite small, and I just didn't think having them described as percentages was useful, and also made the results a little harder to read. AUTHOR RESPONSE MC4. In order to provide proportionately comparable results, percentages were chosen to illustrate trends in the two course participant groups. This metric was selected due to differing sample sizes, making raw n hard to interpret, however to the reviewer’s point, we also felt including the n per category was important; hence we elected to present both to the reader." } ] }, { "id": "72972", "date": "30 Oct 2020", "name": "Ronald J. Heustis", "expertise": [ "Reviewer Expertise Graduate classroom-based learning of scientific concepts as well as academic and professional skills", "the longitudinal development of life sciences graduate trainees", "including their skills and career interests." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 - to both the authors and the editor - for the opportunity to review this article. I recommend indexing this article; I have some minor editing comments (relevant to papers that may be cited and some typographical corrections) but that should not detract from my recommendation which is that this article go through the final stage of indexing.\nSummary and key points:\nThis article summarizes the delivery and evaluation of two \"career exploration\" courses, at two different institutions, accessible to both graduate students and postdocs.\n\nGraduate and postdoc professional development has gained recognition and the field of professionals has grown larger. Published literature describing not only best practices but also the evaluation of those efforts is essential to share information within the community, including both those new to the practice and those looking to enhance existing training and evaluation protocols.\nAs a whole the paper provides a valuable perspective on the value of graduate and postdoc professional development courses, integrating what might be a series of standalone workshops into a structured offering. The authors describe two different courses (abbreviated as PSP and HINAP) and they do a good job distinguishing the models of the classes based on theoretical underpinnings. These are two good examples of formal “credit-bearing” courses and how they can benefit students.\nOverall, the literature base - presented in the Introduction and Discussion - is sound, and includes both theoretical and applied publications as well as guiding policy/\"white papers\" which situate the value of this work and its contributions to the field.\nThe methods used in this work are clearly described, including statistical analysis. The authors explain the connection between these results and the conclusions they draw. It is noteworthy that they make efforts to acknowledge the limits of their sample sizes and also, in concluding, discuss how future projects can move to more experimental approaches to enhance the rigor of the evaluation methods used in this publication.\nThe descriptions of the courses outline the following similarities and differences:\nthere are populations of two different sizes both include more participants who identify as female, both of which include participants from different disciplines, and, both of which include some grad students and some postdocs.\n\nSome of the important findings from the paper:\nParticipants in the courses are less likely, at the end of the course, to be completely “uncertain” about a career of interest.\n\nTrainees learn about more career options, and in some cases, they learn enough to shift interest to something else. Career interest is linked to values and values can be independently identified as a starting point, or be revealed through career exploration.\n\nTrainees get a better sense of the “next steps” including more details on the process of applying for jobs in certain sector and/or what it is like to work in those sectors.\n\nCareer courses can be a good way to help students build their mentor networks.\n\nOne of the very important findings from the results presented here is the observation that participation in these courses increases interest in tenure-track faculty careers. This is a very important point to underscore particularly as institutions grapple with national research funding mandates (as well as ethical priorities) to provide opportunities for trainees to explore careers, while addressing historical/institutional culture and faculty who may not support these goals.\n\nConceptual edits/places for commentary:\nSome aspects that do not necessarily require change, but may benefit from clarification/comment:\nIn the Introduction, the authors mention two references pointing to the fact that 2/3 of postdocs move onto non-tenure track or nonacademic positions. If the goal of this was to note only select examples of this evidence, no change necessary. If the intention was to be comprehensive, I would also include Silva et al. (20161) as this publication similarly shows this proportion of trainees (of those U.S. trained PhDs) moving on to research tenure-track positions.\n\nIn the Introduction, there is a sentence: “This framework is a variation from traditional themes of “career awareness”, “career exploration,” and “career preparation/career immersion” (e.g. Colorado, Massachusetts)”. I believe these are references to the state plans for ILPs mentioned in the follow-up sentences, but this could be clarified before referencing the states.\n\nIn both studies, there is a conspicuous over-representation in the participants who identify as women in the study population. This breakdown by gender is very possible from a convenience sample (i.e. from those who register) but it might be worth commenting on factors that might influence women to pursue this type of opportunity over men. (This might be as simple as reflecting on the proportions in the trainee population.)\n\nFor the PSP course, n=32...over how many years/cohorts? Was it one offering of the 7-week version and one offering of the 17-week version?\n\nFor the PSP course, is there any reason to believe that that 17-week vs 7-week versions of the course might show differential outcomes? I know that given the sample size no conclusions can be drawn here, but for future reference it is worth noting as a consideration. The lengthened immersion may allow for more time for learning consolidation; however, the shorter version is likely more practical and if it accomplishes the same goals/outcomes of an extended course, the 7-week version seems like a recommendable best practice.\n\nFor the PSP course, there is a heavy emphasis on 1st and 2nd year students. This is not explained by the proportion of master's students (13%). Is there a factor that explains why students are so engaged so early in the career exploration? Do certain programs require this course? Published literature, including references in this paper (Fuhrmann et al. 2011 CBE-Life Sciences Education), suggest that students begin considering other careers slightly later…between years 2/3 of doctoral training. So, it is just interesting to note that there is such interest in the first-year students here.\n\nFor the HINAP course, why were career advising staff only included for the post-course surveys?\n\nOverall, the Discussion of the manuscript can be enhanced by comparing the results of this work with those of Branan et al. (20182) which describes a career exploration course for which the evaluation includes learning gains on overlapping outcomes such as understanding and using SMART objectives. This comparison would be more relevant for the HINAP course.\n\nSome questions, in general, for future inquiry:\nIn reading this paper, two things came to mind. These are outside the scope of this paper, but may be future questions as we get more data from similar course implementations. The authors, if they have opinions on this or any relevant data from the course, are of course welcome to comment:\n\nWhen we look at the impact of these courses to influence student career choice by engaging speakers in the different career trajectories, how much does the personality of the presenters, and alignment with the students’ personalities matter? How, too, does the level of satisfaction with their own careers affect presenters' narratives and the influence they can have on the students.\n\nFor these studies, it would be interesting to follow-up with participants to see how the course ends up affecting them after course completion as in longer-term follow-up to find out if they class affected not only self-efficacy but also behavior.\n\nEditing changes:\n\"Biomedical Career Path Familiarity. Using a four-point scale ranging from 1 (Not at all familiar) to 5 (Very familiar), participants were asked to rate how familiar they were with 15 career paths (mirroring NIH BEST Consortium baseline survey; Lenzi et al., 2020).\" This is a either a FIVE-POINT scale or the 5 should be a 4.\n\nIn the PSP discussion, there is a missing phrase in this sentence I believe: “Participants also reported more awareness of how the transferable skills they were developing in the graduate programs and more confidence on career management planning”. I think there should be something like “…the transferable skills they were developing in graduate programs could be applied…” or perhaps just remove the word “how”.\n\nFor the summary of the HINAP course, “Methods. Participants. A total of 148 respondents…completed pre- or post-data over a five year period.” Either “completed” should be “contributed” or “data” should be “surveys”.\n\nThere is a grammatical error in this sentence, from the general Discussion... \"This study evaluated the thematic content and course evaluations for two independently developed, complimentary academic courses career development courses for doctoral and postdoctoral trainees offered at the two distinct institutions.\"\n\n\"Principal Investigator (PI) engagement in mentorship showed no change pre- to post-course, which might be interpreted either as continued PI involvement in mentoring their trainees, irrespective of trainee participation in the course. ~The word \"either\" doesn't fit unless the sentence was meant to be completed with something else.\n\n\"Hence the recommendation to enhance the connections available to graduate and postdoctoral trainees via programmatic partnerships as a whole, which will facilitate departmental and institutional access to professionals across a variety of careers, to expose and connect trainees with.\" ~ This sentence is incomplete or should be re-structured.\n\nIn the Conclusions, “The second institution (HINAP) course focused on examining interests and setting goals using the online career information myIDP”. I believe “information” should be “platform”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7661", "date": "03 Feb 2022", "name": "Rebekah Layton", "role": "Author Response", "response": "Reviewer 2 - Ronald J. Heustis 1 Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA 2 Program in Graduate Education, Harvard Medical School, Boston, MA, USA R2.0. Thank you - to both the authors and the editor - for the opportunity to review this article. I recommend indexing this article; I have some minor editing comments (relevant to papers that may be cited and some typographical corrections) but that should not detract from my recommendation which is that this article go through the final stage of indexing. Summary and key points: This article summarizes the delivery and evaluation of two \"career exploration\" courses, at two different institutions, accessible to both graduate students and postdocs. Graduate and postdoc professional development has gained recognition and the field of professionals has grown larger. Published literature describing not only best practices but also the evaluation of those efforts is essential to share information within the community, including both those new to the practice and those looking to enhance existing training and evaluation protocols. As a whole the paper provides a valuable perspective on the value of graduate and postdoc professional development courses, integrating what might be a series of standalone workshops into a structured offering. The authors describe two different courses (abbreviated as PSP and HINAP) and they do a good job distinguishing the models of the classes based on theoretical underpinnings. These are two good examples of formal \"credit-bearing\" courses and how they can benefit students. Overall, the literature base - presented in the Introduction and Discussion - is sound, and includes both theoretical and applied publications as well as guiding policy/\"white papers\" which situate the value of this work and its contributions to the field. The methods used in this work are clearly described, including statistical analysis. The authors explain the connection between these results and the conclusions they draw. It is noteworthy that they make efforts to acknowledge the limits of their sample sizes and also, in concluding, discuss how future projects can move to more experimental approaches to enhance the rigor of the evaluation methods used in this publication. The descriptions of the courses outline the following similarities and differences: ○ there are populations of two different sizes ○ both include more participants who identify as female, ○ both of which include participants from different disciplines, and, ○ both of which include some grad students and some postdocs. Some of the important findings from the paper: ○ Participants in the courses are less likely, at the end of the course, to be completely \"uncertain\" about a career of interest. ○ Trainees learn about more career options, and in some cases, they learn enough to shift interest to something else. Career interest is linked to values and values can be independently identified as a starting point, or be revealed through career exploration. ○ Trainees get a better sense of the \"next steps\" including more details on the process of applying for jobs in certain sector and/or what it is like to work in those sectors. ○ Career courses can be a good way to help students build their mentor networks. ○ One of the very important findings from the results presented here is the observation that participation in these courses increases interest in tenure-track faculty careers. This is a very important point to underscore particularly as institutions grapple with national research funding mandates (as well as ethical priorities) to provide opportunities for trainees to explore careers, while addressing historical/institutional culture and faculty who may not support these goals. AUTHOR RESPONSE 2.0: Thank you for distilling the key points; we are glad these messages came across effectively in our study. We agree and hope that together these findings demonstrate the value of career courses being offered; show tangible benefits to participants; and alleviate any hesitation of faculty due to potential concerns, given that we demonstrate that offering career exploration and planning opportunities does not have to detract at all from preparing for the professoriate. We posit that taken together, this evidence suggests that career preparation for academic and non-academic pathways can be complementary and mutually beneficial processes, regardless of the career path ultimately chosen. R2.1: Some aspects that do not necessarily require change, but may benefit from clarification/comment: In the Introduction, the authors mention two references pointing to the fact that 2/3 of postdocs move onto non-tenure track or nonacademic positions. If the goal of this was to note only select examples of this evidence, no change necessary. If the intention was to be comprehensive, I would also include Silva et al. (2016) as this publication similarly shows this proportion of trainees (of those U.S. trained PhDs) moving on to research tenure-track positions. AUTHOR RESPONSE 2.1. While this list is not intended to be comprehensive but rather illustrative of recent career trends regarding the professoriate, we have now added, “e.g.,” in text to clarify. Because Silva et al. (2016) explicitly avoids breaking career outcomes down into tenure track, the numbers aren’t directly comparable in this particular context (rather, Silva et al 2016 specifically avoids this classification, and instead restricts to a broad academic/teaching and research careers combined group). Nonetheless, a reference to Silva et al (2016) has been added elsewhere in the manuscript as relevant and we thank the reviewer for noting this relevant contribution to the literature. R2.2: In the Introduction, there is a sentence: \"This framework is a variation from traditional themes of \"career awareness\", \"career exploration,\" and \"career preparation/career immersion\" (e.g. Colorado, Massachusetts)\". I believe these are references to the state plans for ILPs mentioned in the follow-up sentences, but this could be clarified before referencing the states. AUTHOR RESPONSE 2.2. Thank you for pointing this out. While ILP is the national term, most states and programs use their own naming convention such as individual career and academic plan, IDP, etc. We have removed reference to the state-specific examples as we agree that this was confusing. Reviewer 1 had a similar comment. Please see our response to Reviewer 1 for more details (see Author Response 1.MC1). R2.3: In both studies, there is a conspicuous over-representation in the participants who identify as women in the study population. This breakdown by gender is very possible from a convenience sample (i.e. from those who register) but it might be worth commenting on factors that might influence women to pursue this type of opportunity over men. (This might be as simple as reflecting on the proportions in the trainee population.) AUTHOR RESPONSE 2.3. We agree with the reviewer that further elaborating on this point would be helpful—however, the data collected does not allow us to understand the significance of the over-representation of women in these two studies. Hence, we have added a section on Limitations and Future Directions to better address this and call for further investigation. Reviewer 1 also had a similar question, please find additional detail in our author response (see Author Response 1.6). R2.4: For the PSP course, n=32...over how many years/cohorts? Was it one offering of the 7-week version and one offering of the 17-week version? For the PSP course, is there any reason to believe that that 17-week vs 7-week versions of the course might show differential outcomes? I know that given the sample size no conclusions can be drawn here, but for future reference it is worth noting as a consideration. The lengthened immersion may allow for more time for learning consolidation; however, the shorter version is likely more practical and if it accomplishes the same goals/outcomes of an extended course, the 7-week version seems like a recommendable best practice. AUTHOR RESPONSE 2.4. Indeed, preliminary comparisons (see Supplemental Table 1) demonstrated that long versus short courses had comparable outcomes, suggesting that if short courses are as effective as long course might be more efficient to utilize short courses when practicable. Although as mentioned by the reviewer, the sample size was too small to draw robust conclusions and hence this analysis was not included in the primary text, future directions might consider comparing larger samples to further confirm if these initial observations are replicable. Course type/length. As can be seen in Supplemental Table 1 (available in Open Science Framework folder at: https://doi.org/10.17605/OSF.IO/9WRBY), there were gains by both groups in composite skills; in some cases, surprisingly, the mini-course even exhibited a greater gain than the full semester course. Please note that these results should be interpreted with caution due to the small sample size and potential confounds, and should be replicated in larger samples, across career course offerings, and across diverse graduate student and postdoctoral trainee populations. R2.5: For the PSP course, there is a heavy emphasis on 1st and 2nd year students. This is not explained by the proportion of master's students (13%). Is there a factor that explains why students are so engaged so early in the career exploration? Do certain programs require this course? Published literature, including references in this paper (Fuhrmann et al. 2011 CBE-Life Sciences Education), suggest that students begin considering other careers slightly later…between years 2/3 of doctoral training. AUTHOR RESPONSE 2.5. Typically, if enrolled in the course during the early years, students get the benefit of attending and participating, and PIs don't have to pay additional funds; this may be more convenient for both PIs and students. This is because in many programs, including those in this study, after the second year the PI may have to pay additional funds for their students to participate coursework. This could influence students to choose not to sign up for any additional for-credit courses, make them hesitant to ask their PI for funding to participate, and/or may be limited by actual availability of funds from the lab. While interest in career exploration may be higher in later graduate years as the reviewer notes, we infer that the structural barriers to completing coursework may be a likely drivers of early student participation during initial years of graduate coursework as opposed to later in their training. Nonetheless, a valid question is if these financial and perceived barriers could be removed, would there be a greater demand in later years -- we suspect that indeed that could alter the demographic make-of participants, but future studies would need to test this hypothesis. Furthermore, another question that may arise is whether the course would be comparatively more impactful in later years of training. This relates to the question of how stage of training may influence participation and career development, and is now addressed in Limitations and Future Directions (see Author Response 1.7 & 1.8 for more on stages of training). R2.6: For the HINAP course, why were career advising staff only included for the post-course surveys? AUTHOR RESPONSE 2.6. One limitation to our data is that Program Directors did not initially include staff mentorship as an option in the initial questionnaire, but it was later added. For full transparency and to be able to address this limitation, the data is presented with and without mentors and staff in Table 5. Importantly, in both cases, we see a positive shift and increase in the participant's professional mentors cited. R2.7: Overall, the Discussion of the manuscript can be enhanced by comparing the results of this work with those of Branan et al. (2018) which describes a career exploration course for which the evaluation includes learning gains on overlapping outcomes such as understanding and using SMART objectives. This comparison would be more relevant for the HINAP course. AUTHOR RESPONSE 2.7. The reviewer brings up an important point; the course described in the manuscript Branan et al (2018) offered six weeks of training, including self-assessment and career exploration, before the exercise of establishing (SMART) goals. We agree that a similar addition would be beneficial for HINAP course. Therefore, we have added a citation for Branan and colleagues’ previous work, and included the following sentence in the Recommendations section, “For instance, the emphasis placed on self-assessment and career exploration before making career choices and setting SMART goals could benefit HINAP (e.g., Branan et al, 2018).” R2 Comments: In reading this paper, two things came to mind. These are outside the scope of this paper, but may be future questions as we get more data from similar course implementations. The authors, if they have opinions on this or any relevant data from the course, are of course welcome to comment: R2.C1: When we look at the impact of these courses to influence student career choice by engaging speakers in the different career trajectories, how much does the personality of the presenters, and alignment with the students' personalities matter? How, too, does the level of satisfaction with their own careers affect presenters' narratives and the influence they can have on the students. AUTHOR RESPONSE 2.C1: The reviewer raises an important point; there could certainly be influences of the speakers themselves based on their own career satisfaction and personality styles. We suspect that a sample bias occurs whereas primarily only speakers who are relatively happy with their career trajectory and outcomes agree to present for such courses, however this may vary between people or could be an incorrect assumption and should be recorded and tested in future studies as it may provide interesting context for how speaker choices by course organizers may influence student receptivity to those particular careers. Personality matches, as well as other characteristics of whether a person can envision themselves in that role (including race/ethnicity, gender, LGBTQIA+ identification, religion, international status, first generation status, and more) may influence course participants’ receptivity to those particular career paths as well. We agree that this would be an important aspect for course directors to consider in future career course planning and interventions, and that evidence-based data to evaluate these influences would be incredibly important to gather. While our data does not allow us to address this, we have added the following comment to Limitations and Future Directions, “Similarly, the influence of speaker characteristics may also differentially facilitate course participants being able to imagine themselves in that particular career path depending on how well they connect with or identify specific speakers. These characteristics could include social identity groups, different background and experiences, perceived personality similarities, as well as different levels of enthusiasm about the speakers’ own specific career pathway.” R2.C2: For these studies, it would be interesting to follow-up with participants to see how the course ends up affecting them after course completion as in longer-term follow-up to find out if they class affected not only self-efficacy but also behavior. AUTHOR RESPONSE 2.C2.1. We agree that the true value of the course may not be evident until well into the trainees' future careers, or at the very least after their initial first career choice. This would be a meaningful and important contribution to the literature. For the current courses, the number of trainees that had graduated or departed the university was a small subsegment of the sample, leading to a small sample size as many trainees were still affiliated with their respective institutions, and hence we were not able to examine this longer-term outcome. However, we have added the following to Future Directions to acknowledge this important point, “Finally, longitudinal studies could examine career outcomes such as career choice (first placement) and career satisfaction, which would add value to the literature.” R2.C2.2. \"Biomedical Career Path Familiarity. Using a four-point scale ranging from 1 (Not at all familiar) to 5 (Very familiar), participants were asked to rate how familiar they were with 15 career paths (mirroring NIH BEST Consortium baseline survey; Lenzi et al., 2020).\" AUTHOR RESPONSE 2.C2.2. This is a a five-point scale, and has been corrected accordingly. R2.C2.3. In the PSP discussion, there is a missing phrase in this sentence I believe: \"Participants also reported more awareness of how the transferable skills they were developing in the graduate programs and more confidence on career management planning\". I think there should be something like \"…the transferable skills they were developing in graduate programs could be applied…\" or perhaps just remove the word \"how\". AUTHOR RESPONSE 2.C2.3. The word “how” has been removed. R2.C2.4. For the summary of the HINAP course, \"Methods. Participants. A total of 148 respondents…completed pre- or post-data over a five year period.\" Either \"completed\" should be \"contributed\" or \"data\" should be \"surveys\". AUTHOR RESPONSE 2.C2.4. This has been updated to “pre- and post- course surveys.” R2.C2.5. There is a grammatical error in this sentence, from the general Discussion... \"This study evaluated the thematic content and course evaluations for two independently developed, complimentary academic courses career development courses for doctoral and postdoctoral trainees offered at the two distinct institutions.\" AUTHOR RESPONSE 2.C2.5. Thank you this has been corrected to read: “This study evaluated the thematic content and course evaluations for two independently developed, complimentary academic career development courses for doctoral and postdoctoral trainees offered at the two distinct institutions.” R2.C2.6.\"Principal Investigator (PI) engagement in mentorship showed no change pre- to postcourse, which might be interpreted either as continued PI involvement in mentoring their trainees, irrespective of trainee participation in the course. ~The word \"either\" doesn't fit unless the sentence was meant to be completed with something else. AUTHOR RESPONSE 2.C2.6. Thank you, the word “either” has been removed. R2.C2.6. \"Hence the recommendation to enhance the connections available to graduate and postdoctoral trainees via programmatic partnerships as a whole, which will facilitate departmental and institutional access to professionals across a variety of careers, to expose and connect trainees with.\" ~ This sentence is incomplete or should be re-structured. AUTHOR RESPONSE 2.C2.7. Thank you we have replaced this sentence with the following revision: “Therefore it is important to programmatically enhance the connections available to graduate and postdoctoral trainees via partnerships, facilitating departmental and institutional access to professionals across a variety of careers.” R2.C2.7. In the Conclusions, \"The second institution (HINAP) course focused on examining interests and setting goals using the online career information myIDP\". I believe \"information\" should be \"platform\". AUTHOR RESPONSE 2.C2.8. Thank you for noting this, “information” has been replaced with “platform.”" } ] } ]	1	https://f1000research.com/articles/9-1230
https://f1000research.com/articles/11-141/v1	03 Feb 22	{ "type": "Research Article", "title": "A Study of Catastrophic Health Expenditures in India - Evidence from nationally representative survey data: 2014-2018", "authors": [ "Shyamkumar Sriram", "Muayad Albadrani", "Muayad Albadrani" ], "abstract": "Abstract\nBackground: India is taking steps to provide Universal Health Coverage (UHC). Out-of-pocket (OOP) health care payment is the most important mechanism for health care payment in India. This study aims to investigate the effect of OOP health care payments on catastrophic health expenditures (CHE). Methods: Data from the National Sample Survey Organization, Social Consumption in Health 2014 and 2018 are used to investigate the effect of OOP health expenditure on household welfare in India. Three aspects of catastrophic expenditure were analyzed in this paper: (i) incidence and intensity of ‘catastrophic’ health expenditure, (ii) socioeconomic inequality in catastrophic health expenditures, and (iii) factors affecting catastrophic health expenditures. Results: The odds of incidence and intensity of CHE were higher for the poorer households. Using the logistic regression model, it was observed that the odds of incidence of CHE was higher among the households with at least one child aged less than 5 years, one elderly person, one secondary educated female member, and if at least one member in the household used a private healthcare facility for treatment. The multiple regression model showed that the intensity of CHE was higher among households with members having chronic illness, and if members had higher duration of stay in the hospital. Subsidizing healthcare to the households having elderly members and children is necessary to reduce CHE. Conclusion: Expanding health insurance coverage, increasing coverage limits, and inclusion of coverage for outpatient and preventive services are vital to protect households. Strengthening public primary health infrastructure and setting up a regulatory organization to establish policies and conduct regular audits to ensure that private hospitals do not increase hospitalizations and the duration of stay is necessary.", "keywords": [ "financial protection", "out-of-pocket health expenditure", "catastrophic health expenditures", "universal health coverage" ], "content": "Introduction\n\nGoal 3 of the United Nation’s Sustainable Development agenda has the specific goal to provide universal health coverage (UHC) to its population and to improve financial protection. UHC includes securing access to quality healthcare and safe, affordable medicines and vaccines for everyone (Saksena et al., 2014). Resolution 58.33 of the World Health Assembly recommends that all WHO member states should provide UHC to their entire population and protect households from catastrophic health expenditures (CHE) (Obermann et al., 2018). Any household out-of-pocket (OOP) health spending that exceeds a certain proportion of household’s financial ability is defined as CHE (Xu et al., 2003). Globally around 100 countries have achieved UHC or initiated reforms towards UHC (Obama, 2008; Summers, 2015). Even though most countries are striving to enable their citizens to obtain the healthcare they need without financial barriers, 808 million people still experience CHE each year (Wagstaff et al., 2018). More than 90% of the people experiencing CHE live in low-income countries (Xu et al., 2003). Financial protection needed to a population depends on their dependence on OOP health expenditure for paying for healthcare (Xu et al., 2003). Dependence of the households on OOP payments for obtaining healthcare escalates the financial burden of the households (Amaya et al., 2011; Wagstaff et al., 2003; Xu et al., 2003). With OOP health expenditures constituting around 62.6% of total health expenditures, India ranks third in the Southeast Asia region among countries with high OOP health expenditure (Balarajan et al. 2011; Hooda, 2017; Sriram 2018).\n\nMany studies have examined the health expenditures on specific diseases such as diabetes, tuberculosis, cancer, injuries etc., but the problem was that most of these studies were done in small geographical areas of the country and their representativeness for the whole nation was limited (Binnendijk et al., 2012; Yesudian et al., 2014; Rao et al., 2011; Prinja et al., 2015; Muniyandi et al., 2005; Ramachandran et al., 2007). Some studies have examined the determinants of OOP health expenditures for outpatient care in a few districts of India for certain age groups (Brinda et al., 2012; Gupta et al., 2016). Also, other studies have used different National Sample Survey Organization (NSSO) datasets and other nationally available data like National Family Health Survey (NFHS), etc. to study disease-specific OOP health expenditures for hospitalizations (Kastor & Mohant, 2018). Studies have looked at OOP health expenditures due to non-communicable diseases (NCDs) (Tripathy et al., 2016), burden of OOP payments due to medicines (Selvaraj et al., 2018), OOP health expenditure for maternal care (Mohanty & Kastor, 2017), OOP health expenditure for accidental injury (Pradhan et al., 2017), but they did not address the specific research questions related to CHE in general and factors affecting incidence and depth or gap of CHE.\n\nThe primary objective of the study is to detect the features of households, health conditions, and health delivery system issues that make people prone to CHE. In particular, the study will examine the association of households’ demographic characteristics, social structure, and healthcare utilization that appear to be associated with relatively high levels of expenditure and also quantify the burden of OOP health expenditures and CHE. In this research, we used the data from two surveys, the 2014 and 2018 NSSO data to assess the level of financial protection in India (NSSO, 2014). To measure the effect of CHE on households, we estimate (i) incidence, intensity, and inequality in CHE in India (ii) incidence and intensity of CHE across different states in India (iii) various aspects influencing the incidence and intensity of CHE in India.\n\nIndia is taking steps to provide UHC to its citizens. By identifying the incidence, intensity, socioeconomic inequalities in CHE, this study helps the central government provide an appropriate higher budgetary allocation for the groups that have higher OOP health expenditures and helps in designing health insurance programs that benefit the poor. This research will help in reducing catastrophic spending in India. Andersen’s Behavioral Model of Healthcare Utilization as shown in Figure 1 will be used to guide this research (Andersen, 1995). The Andersen model examines the predisposing, enabling, need and healthcare utilization characteristics.\n\n\nData\n\nThe data from the NSSO of the Government of India were used for the study. The study only used secondary data analysis obtained from the Government of India. Only deidentified data has been provided by the Government of India. No ethical approval was required since there is no human participation in the study. Social Consumption (Health), NSS 71st Round and NSS 75th Round were used for this analysis. Both the surveys covered whole of the Indian Union. The surveys collected data from 65,932 randomly selected households (NSS 71st Round) and 113,823 households (NSS 75th Round) The data for the NSS 71st round survey were obtained from January to June 2014 and the data for the NSS 75th round were collected between July 2017 and June 2018 (NSSO, 2014).\n\n\nMethods\n\nThe incidence of CHE was calculated from the proportion of OOP healthcare payments which exceed a certain threshold in relation to the household consumption expenditure (Wagstaff et al., 2003). In this research, the OOP health expenditure is compared with the household consumption expenditure, and it is assumed that a household experienced CHE if health expenditure exceeds the 10% threshold level. Catastrophic payment headcount informs the proportion/number of households affected by CHE i.e., the number of households who are experiencing an OOP healthcare expenditure above 10% of household consumption expenditure.\n\nCatastrophic payment headcount is given by the formula:\n\nHC is the catastrophic payment headcount. The indicator E=1 is defined when Ti/Xi >Z and zero otherwise. Here Z is 0.10. T is the household OOP health expenditure; X is the total household consumption expenditure and N is the sample size. The theoretical minimum and maximum values of catastrophic payment headcount are 0% and 100% respectively. The CHE incidence (headcount) does not indicate the degree to which the household’s CHE exceed the threshold value, thus the CHE intensity (overshoot) has also been estimated. The intensity (overshoot) of the CHE is the average degree when the household OOP health expenditures as a proportion of the household consumption expenditure exceeds the pre-specified thresholds (10%).\n\nAverage catastrophic excess (O) measures this intensity of CHE, and it is given by the formula below:\n\nOi is the excess or overshoot and it is calculated by the formula, Oi=Ei [(Ti/xi)-Z]. Z is the threshold budget share. The minimum and maximum value of catastrophic payment gap is 0% and 90% respectively when the threshold value is fixed at 0.10.\n\nThe measures of CHE are insensitive to household economic conditions and thus do not identify whether the poor or rich households exceed the threshold more (O’Donnell et al., 2008). Many policymakers will consider it a significant problem if the poorer households exceed the threshold level compared to the richer households. Wagstaff et al. recommend the calculation of concentration indices to separate the association of CHE with socio-economic status (Wagstaff et al., 1989). Concentration indices are used to detect the presence of socioeconomic inequality in any health sector variable and whether it is more marked in one group than another (Kakwani 1977; Kakwani et al., 1997; Takayama and Nanack, 1983; Wagstaff et al., 1989).\n\nTo investigate the effects of different factors on the incidence of CHE, the logistic regression model is used. A dichotomous variable for CHE is created with 0 for not incurring catastrophic health expenditures and 1 for incurring catastrophic health expenditures. Thus, the dichotomous variable created for CHE will serve as the dependent variable for the logistic regression model. Among the households which incurred CHE, intensity of CHE was calculated, and multiple regression model was used to identify factors affecting intensity levels. The dependent variable is the catastrophic payment gap, and the independent variables included various characteristics of the individuals, households and health facility. Globally many studies have those specific household characteristics such as location, size of the household, utilization of private health facilities, health insurance coverage, presence of chronic illnesses, hospitalizations, presence of elderly and children in the household (Bhojani et al., 2012; Brinda et al., 2014; Dwivedi & Pradhan 2017; Mondal et al., 2014; Srivastava et al., 2009; Leive & Xu., 2008).\n\n\nResults\n\nDescriptive statistics presented in Table 1. There were 65,932 households in the sample in the 2014 data and 113,823 households in the 2018 data. 33% of the households (2014) and 25% of households (2018) have at least one child aged 5 years and less; 26.87% households (2014) and 22.92% households (2018) have at least one elderly person. The proportion of households located in rural and urban areas are almost same in 2014 and 2018. About 9.98% of the households (2014) and 15.40% of households (2018) have members who used a private hospital.\n\n* Sample size N = 65,932 (2014) and N= 113,823 (2018).\n\nTable 2 shows that the CHE incidence was 10.94% in 2014 and 16.51% in 2018. Incidence of CHE was 64.57% (2014) and 43.99% (2018) among households that used a private hospital. Table 3 shows that the mean positive overshoot indicates that on average, the OOP was 35.94% (2014) and 34.08% (2018).\n\nFigure 2 and Figure 3a show the incidence of CHE for the states in India for the years 2014 and 2018. Figure 2 and Figure 3b show the intensity of CHE for the states in India for the years 2014 and 2018. Figure 4 shows the change in CHE between 2014 to 2018 across the different states. In 2018, Kerala (33.77%) has the highest incidence of CHE, while Meghalaya (1.83%) has the lowest incidence of CHE. In 2014, Kerala (15.71%) has the highest incidence of CHE, while Chandigarh (2.80%) has the lowest incidence of CHE. In 2018, Arunachal Pradesh (53.86%) has the highest intensity of CHE, while Daman and Diu (6.45%) has the lowest intensity of CHE. In 2014, Chhattisgarh (47.38%) has the highest intensity of CHE, while Daman and Diu (10.55%) has the lowest intensity of CHE. Kerala (18.06%) experienced the highest increase in incidence of CHE from 2014 to 2018, while Goa (5.85%) experienced a decline in incidence of CHE from 2014 to 2018. Manipur (24.75%) experienced the highest increase in intensity of CHE from 2014 to 2018, while Uttaranchal (24.67%) experienced a decline in intensity of CHE from 2014 to 2018. In 2014, households in the richest expenditure quintile have the highest incidence of CHE, while in 2018, households all the income expenditure quintiles incur almost more or less same incidence of CHE as shown in Table 4.\n\nTable 5 shows that both in 2014 and 2018, it was observed that the odds of experiencing CHE was higher among households with children, elderly person, and utilization of a private hospital. Urban households had lower probability of experiencing incidence of CHE, and households from all other expenditure quintiles also had lesser odds of incurring CHE compared to households in the poorest quintile both in 2014 and 2018. The likelihood of incidence of CHE increased with the increase in duration of stay in the hospital, with the highest odds being for the households who had members who stayed for more than 20 days in a hospital. Also, the presence of chronic illness among members in the household increased odds of CHE. Health insurance coverage in the household reduced the likelihood of CHE incidence. Table 6 shows that both in 2014 and 2018, households’ children aged less than 5 years, members being covered by health insurance, and not belonging to the poorest expenditure quintile had lower intensity of CHE.\n\n\nDiscussion\n\nIn 2014, among the households which experienced CHE, the mean positive overshoot indicates that on average, the OOP health expenditures was 35.94% higher, but the overshoot decreased to 34.08% in 2018. This shows that although the intensity is very high among the households experiencing CHE, but it is decreasing. Our study showed that both in 2014 and 2018, a higher odds of incidence of CHE among the households with children, elderly people and those who used a private hospital for treatment. This was consistent with literature which showed that households which consisted of members at extremes of age (Mohanty et al., 2014; da Silva et al., 2015), members utilization of private health facility (Alam & Mahal 2014; Saksena et al., 2012; Somkotra & Lagrada, 2009) had higher OOP and CHE. The likelihood of incidence and intensity of CHE in our study increased progressively with the increase in duration of hospital stay. However, the effect of duration of hospitalization on incidence of CHE was much lower in 2018 compared to 2014, but the intensity of CHE was much higher in 2018 compared to 2014. This shows that health insurance and other health financing protection mechanisms may have been successful in reducing the number of households being pushed to experience CHE, but among the households that experienced CHE, the burden (overshoot) has increased greatly over this time-period. A World Bank study showed that hospitalizations are the major drivers of OOP health expenditures (McIntyre et al., 2006). Also, the presence of chronic illness among members in the household increased odds of CHE incidence and also increased the intensity among households experiencing CHE. Similar results were found in India (Mondal et al., 2014), Bangladesh (Molla et al., 2017), and China (You & Kobayashi, 2011) that showed that chronic illness is an important determinant for experiencing CHE. Our study showed that the presence of health insurance coverage among members in the household reduced the likelihood of CHE incidence and even among the households that experienced CHE, the intensity was lesser for households that had health insurance coverage. Other studies from India (Fan et al., 2012), Indonesia (Aji et al., 2013), Laos (Alkenbrack & Lindelow; 2015), and Vietnam (Sepehri 2013) supported this finding of the protective effect of health insurance from CHE.\n\nThe regression results show that the households from all other expenditure quintiles had lesser odds of incurring CHE compared to poorest households. Among the households that experienced CHE, the intensity was also highest among the poorest households. For the poorer households, high level of intensity or overshoot may be due to low level of absolute income. It is expected that the poor people are more prone to experience CHE, since they have lower level of income and any expenditure that incur for healthcare will easily make it “catastrophic” since the proportion of the health expenditure will become relatively high for them because of low total consumption expenditure (low value of denominator). Thus, poorest people have higher risk of facing CHE even with a relatively small adverse health event. Globally there is mixed evidence on the relationship between socio-economic status and CHE. These results are similar with the findings from research done in India (Bhojani et al., 2012), Thailand, Paraguay, and Burkina Faso (Makinen et al., 2000) which showed that low-income households were associated with a higher likelihood of CHE. Other studies in Nigeria, Namibia, Albania, Kenya, Bangladesh, and India show that poorer households have lower absolute OOP health expenditures compared to richer individuals and households, but the relative proportion of OOP health expenditures to non-food household expenditures was higher in poor households (Chuma & Maina 2012; Gustafsson-Wright et al., 2011; Hotchkiss et al., 2005; Karan et al., 2014; Onwujekwe et al., 2014; Rahman et al., 2013). However, studies from 13 low-income Asian countries (O'Donnell O et al. 2008), Sri Lanka, South Africa and Guatemala (Makinen et al., 2000) showed that richer households spent more on OOP health expenditures and also enjoyed a wide range of services. There is an increase in both the incidence and intensity of CHE with higher duration of hospital stay. Higher duration of hospital stay increases the chance of experiencing CHE. When the higher health expenditures are inadequately covered by health insurance, OOP health expenditures may become catastrophic for many households.\n\nThere are wide differences among states of India in CHE experienced by them. Epidemiological transition and economic growth also lead to an increase in CHE. Higher economic growth leads to an increase in demand for healthcare services and increase in technology used in healthcare delivery. For example, Kerala being an economically advanced state experienced higher incidence of CHE. States like Meghalaya and Nagaland which are economically poor experienced lower incidence of CHE. In the developed states of India, the higher CHE may be due to increased awareness of health benefits and increased utilization of private healthcare facilities over the free public health facilities. The epidemiological and disease burden in a state also determines the healthcare utilization, type of healthcare used, and the volume of healthcare services used in the state. For example, some economically better states may suffer from higher burden of non-communicable diseases, while in contrary some of the poorer states may suffer from maternal and child health problems and infectious diseases. The type of disease experienced by the states directly determines the health expenditures experienced by them (Wagstaff et al., 2018; Kea et al., 2011; Kien et al., 2016).\n\nAlso, in India there is a very poor non-communicable disease service provision in the public sector, and this makes more people choose the private sector hospitals for non-communicable disease treatment which increases their OOP costs when not adequately covered by any health insurance. Different state health insurance programs increased the usage of more private healthcare facilities by increasing the access to them, they have not actually protected the households from the financial burden that arises due to the usage of private health facilities (Karan et al., 2017; Selvaraj & Karan, 2012; Ranjan et al., 2018).\n\nIn conclusion, this research helps policymakers to design health insurance programs to better serve the population. Health insurance benefit packages and coverage limits may be adjusted based on the income levels of poor households with the poorest group receiving the highest level of protection. This type of targeting is also difficult to implement in practice, but it is not impossible with help from community organizations representing the poor and extremely poor households. Households with children less than 5 years and elderly more than 60 years have higher CHE incidence. Children and elderly are the vulnerable age groups who are prone to higher level of health risks. They have higher healthcare utilizations and thus experience higher healthcare expenditures which make the expenditure levels catastrophic in many cases. This implies that policymakers should also consider age as an important factor for health insurance coverage. Health insurance coverage limits in India are restricted and are not adequate especially when the patients stay for longer duration in the hospitals. Thus, the coverage limits for hospital insurance needs to be increased to protect households from CHE. Also, better cost regulation of the private sector hospitals must be done. Chronic illness increases both CHE incidence and intensity. Steps should be taken for early diagnosis and treatment, to reduce the severity of illness, reduce the cost of services, and implementation of better approaches to treat them in the ambulatory settings. This research will help in developing policies to reduce OOP health expenditures in India.\n\n\nAuthor contributions\n\nSS was involved in Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing. MA was involved in Writing – Original Draft Preparation, Writing – Review & Editing.\n\n\nData availability\n\nThe data from the National Sample Survey Organization (NSSO) of the Government of India were used for the study. Social Consumption (Health), NSS 71st Round for 2014 and NSS 75th Round for 2018 were used for this analysis. Both the surveys covered whole of the Indian Union. Data can be obtained from the Government of India or from the corresponding author by reasonable request.", "appendix": "Acknowledgements\n\nThe authors would like to give their sincere thanks to the Government of India for providing the data for the study.\n\n\nReferences\n\nAji B, De Allegri M, Souares A, et al.: The impact of health insurance programs on out-of-pocket expenditures in Indonesia: an increase or a decrease?. Int. J. Environ. Res. Public Health. 2013; 10(7): 2995–3013. 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Publisher Full Text\n\nWagstaff A, van Doorslaer E : Catastrophe and impoverishment in paying for health care: with applications to Vietnam 1993-1998. Health Econ. 2003; 12(11): 921–933. PubMed Abstract \| Publisher Full Text\n\nXu K, Evans DB, Kawabata K, et al.: Household catastrophic health expenditure: a multicountry analysis. Lancet (London, England). 2003; 362(9378): 111–117. PubMed Abstract \| Publisher Full Text\n\nYesudian CAK, Grepstad M, Visintin E, et al.: The economic burden of diabetes in India: a review of the literature. Glob. Health. 2014; 10: 80. PubMed Abstract \| Publisher Full Text\n\nYou X, Kobayashi Y: Determinants of out-of-pocket health expenditure in China: analysis using China Health and Nutrition Survey data. Appl. Health Econ. Health Policy. 2011; 9(1): 39–49. Publisher Full Text" }	[ { "id": "122493", "date": "17 Feb 2022", "name": "Harsh D. Shah", "expertise": [ "Reviewer Expertise Public Health Research", "Operational Research", "Diseases Outbreaks", "Maternal and Child Health", "Epidemiology", "Program Evaluation", "Health Policy and Financing", "Health Economics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe countries should design or customize their health insurance schemes based on pieces of evidence generated through country-specific research studies. The present article has provided key focus areas with stratification of incidence and intensity of catastrophic health expenditure (CHE) of the specific sections of the population and identified the areas where public insurance schemes can concentrate their expansion of existing interventions.\n\nThe article fulfills the criteria for bringing new information, built on replicable methodology, covers the results as intended in objectives and fits in the criteria for research indexing approval.\n\nIs the work clearly and accurately presented and does it cite the current literature? - Partly. The article has supportive references to direct the study findings, but I cannot find references published after the year 2019. The literature search options should be expanded with recent years. The present study does specify the incidence and intensity of CHE with comparison among different strata with statistical analysis through a regression model. The other pieces of literature have not reflected on the same objective and this way, the article shows its uniqueness.\n\nAbstract Section of Conclusion: \"... conduct regular audits to ensure that private hospitals do not increase hospitalizations and the duration of stay is necessary.\" : Avoid the statement without provision of references.\nAre the conclusions drawn adequately supported by the results? Partly. \"Also, in India there is a very poor non-communicable disease service provision in the public sector, and this makes more people choose the private sector hospitals for non-communicable disease treatment which increases their OOP costs when not adequately covered by any health insurance\" - Reference is required if it is not a study finding.\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": "122490", "date": "12 Apr 2022", "name": "Shabana Tharkar", "expertise": [ "Reviewer Expertise Non communicable disease epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written. The author must be applauded for their magnanimous efforts. The study provides useful information to the public health officials and lawmakers in formulating or revising policies and programs incorporating those identified at high risk. The study identifies the determinants of catastrophic health expenditure suggesting the type of population that is most susceptible. The insurance schemes whether public or private can apply these findings to include the vulnerable households in their coverage. Dissemination of the findings may prove valuable to the Health Ministry in initiating relevant programs for the betterment of the vulnerable.\nMinor change required:\nThe first line of the fourth paragraph, under discussion section - “Also, in India there is a very poor non-communicable disease service provision in the public sector, and this makes more people choose the private sector hospitals for non-communicable disease treatment which increases their OOP costs when not adequately covered by any health insurance.” must be reframed in a more polite language and supported by evidence.\nOtherwise the manuscript can be indexed as such. Thank you.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-141
https://f1000research.com/articles/11-140/v1	03 Feb 22	{ "type": "Review", "title": "Food supplements to complement brain functioning: the benefits of a combination of magnesium, folic acid, omega-3 fatty acids and vitamin E", "authors": [ "Rita Businaro" ], "abstract": "Diet and nutrition play a fundamental role not only in human body composition and in physiology, but have also relevant effects on mood, mental well-being and cognitive performance. In particular, the preservation of mental well-being through a healthy lifestyle, including a well-balanced diet and, in case, through the intake of specific food supplements, is of particular relevance in the perspective of global human ageing, as the brain is affected significantly by a persistent presence of stress factors. Due to the increasing burden of mental and neurological disorders and to the universality of food as a modifiable risk factor, even limited improvements in nutritional habits may translate to a considerable rise of well-being and mental health in the global population. Moreover, the use of targeted, well-balanced food supplements aiming to support the mental health and well-being will probably represent a relevant tool in future decades, together with an increased awareness of the importance of nutrition, also considering the COVID-19 pandemic and the related stressful events and limitations we are still experiencing at global level. The aim of this review is to summarize the experimental and clinical data reported in the literature concerning the beneficial effects of a subset of micro- and macronutrients contained both in food and in supplements, namely magnesium, folic acid, docosahexaenoic acid, eicosapentaenoic acid, and alpha-tocopherol, on a series of disorders, including stress, anxiety, low sleep quality, and low cognitive performance.", "keywords": [ "folic acid", "eicosapentaenoic acid", "alpha-tocopherol", "docosahexaenoic acid", "magnesium", "mental well-being", "food supplements", "stress" ], "content": "Introduction\n\nAccording to a definition of the World Health Organization (WHO), mental health is an integral and essential part of health, and is more than just the absence of mental disorders or disabilities. In 2018, the mental health has been defined as a condition of well-being where subjects realize their own abilities, can cope with the normal daily stress, work productively and can contribute to their community (https://www.who.int/news-room/fact-sheets/detail/mental-health-strengthening-our-response). Mental health is at the basis of our ability to interact with each other, think, emote, and poor mental health is associated, among others, with unhealthy lifestyle.\n\nIt is expected that the burden of mood-related disorders and stress-induced cognitive impairment will continue to rise globally in the next years. Changes in lifestyle induced by stress can cause overnutrition and reduced physical activity, possibly leading to obesity and metabolic syndrome; indeed stress is known as one of the main inducers of obesity development and visceral fat, that in turn underlie a faster aging process.1 Alterations in energy expenditure balance disrupt the crosstalk between metabolism and immune system, causing the activation of an immune response and the development of a systemic inflammation with a low-grade, which is one of the main risk factors for the development of depression as well as cognitive impairment.2 According to Straub et al.,3 neuroendocrine pathways are implicated in the regulation of energy: inflammation induces higher levels of cortisol in the serum, by stimulating the hypothalamic-pituitary-adrenal axis (HPA) and the sympathetic nervous system (SNS), leading to a sickness behavior, with a marked reduction of gut, muscle and brain activity. The elevated levels of proinflammatory cytokines observed in systemic inflammation target brain cells binding to specific cytokine receptors expressed on glial cells and neurons thereby directly affecting the brain function, increasing the risk to develop depression. A prolonged exposure to cytokines with a proinflammatory activity impairs synaptic plasticity, contributing to mood alterations as well as to cognitive disorders.4\n\nAccording to WHO, neurological and mental disorders, as well as substance abuse will represent 10% of the global burden of disease and 30% of burden of non-fatal disease (https://www.who.int/news-room/facts-in-pictures/detail/mental-health). On this basis, the protection, restoration and promotion of mental health can be considered a vital concern of subjects, and of worldwide societies and communities. There is a strong need for actions in promotion of mental health to improve psychological well-being (https://www.who.int/news-room/fact-sheets/detail/mental-health-strengthening-our-response).\n\nThis narrative review aims to summarize the experimental and clinical data reported in the literature concerning the beneficial effects of a subset of micro- and macronutrients, namely magnesium, folic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha-tocopherol, on a series of disorders, including stress, anxiety, low sleep quality, and low cognitive performance.\n\n\nThe role of nutrition in mental health and well-being\n\nBrain function, structure and composition are linked to the intake and availability of appropriate micro- and macronutrients, as most essential minerals and vitamins are not produced by the body and should be taken through food. Genetic conditions, medical therapies, inadequate food intake, and systemic diseases can lead to deficiencies of particular nutrients.5 The impact on brain function of food intake and quality makes the diet a modifiable variable to target mental health, mood and cognitive performance. For example, vitamins and minerals play key roles in the body, e.g. in signal transduction or as cofactors in a number of enzymatic reactions. Nutritional excess or deficiencies of some minerals and vitamins can deeply affect the peripheral and central nervous systems from the early development to the adulthood.5 The composition of the diet directly affects also endogenous neuropeptides, neurotransmitters, gut hormones, and the gut microbiota.6,7\n\nDuring the last years, there has been a greater number of epidemiological studies analyzing the link between mental condition and dietary patterns. Longitudinal and cross-sectional studies revealed that the adoption of a Western diet or a diet based on highly processed food proportionally increases the risk of development of psychiatric symptoms, such as anxiety and depression. Conversely, the adoption of a Mediterranean-style diet increased the protection from the development of a mental disorder.8,9\n\nIn several studies, the onset of psychiatric symptoms has been shown to be preceded by a particular dietary pattern. Among them, “SMILES”, a single-blind, parallel-group, 12-week, randomized controlled trial, revealed remarkable effects after 3 months of a dietary intervention on depression of moderate-to-severe grade, with a greater improvement of symptomatology and remission achieved in 32% of participants in the intervention study group.10 The beneficial effects of the Mediterranean-style diet on depression have been confirmed by more recent randomized controlled trials, including the HELFIMED trial (where the Mediterranean-style diet was supplemented with fish oil)11 and the PREDI_DEP trial (where the Mediterranean-style diet was supplemented with extra-virgin olive oil).12 In contrast, supplementation with multi-nutrients in the randomized controlled trial MooDFOOD did not affect the persistence of major depression in subclinical and overweight depressed subjects.13 A study performed on the data of adult subjects from the 9-year follow-up of the Depression and Anxiety Netherlands Study showed a dose-response association between the severity of chronic depression or anxiety disorders and a poorer diet quality in all study participants.14 Overall, the recent findings from clinical trials seem to support the use of a complex of care measures including first line pharmacological and psychological treatments, as well as lifestyle interventions (e.g. improvement of diet quality, addressing smoking cessation, increase of physical activity) may have a greater effect on depression.15 With reference to sleep disorders, and in particular to insomnia, a study has been recently performed on nursing university students to determine the prevalence of sleep disorders and to evaluate in which way eating habits, health status, lifestyle and students’ performance are related to daytime and night-time symptoms of interrupted sleep. The InSOMNIA study revealed a high prevalence of sleep disturbances associated with low adherence to Mediterranean diet.16 The compliance with a Mediterranean-style diet has been associated with lower risk for insomnia and better sleep quality in other recent studies not only in young students, but also in older adults, without gender differences17 and either toward indirect effects or a direct effect on health through a weight improvement.18\n\nMany complex mechanisms are at the basis of the impact of nutrition on the brain and on mental well-being. Recent research has focused in particular on the hippocampus, a region of the brain associated with creativity, memory, learning and mood. The physiological process of aging includes structural changes in the brain where the pattern and rate of hippocampal atrophy is responsible for cognitive impairment and mood disorders often observed in the elderly. An annual 0.2% loss in total brain volume begins in subjects from the age of 35 years.19 The intake of omega-3 polyunsaturated fatty acids (PUFAs) and fish consumption may affect brain morphology changes, as suggested by a randomized controlled trial testing the omega-3 PUFA supplementation in mild cognitively impaired subjects and in cognitively intact individuals. The trials showed that the memory domain of cognitive functions was positively affected by PUFA supplementation.20 The hippocampus is especially modulated by dietary patterns, possibly due to its metabolism and its ability to undergo neurogenesis also during adulthood, well beyond the gestational period. The hippocampus neurogenesis is related to mood and cognition. Consequently, dietary intervention on hippocampal neurogenesis modulation has been considered as a target or a tool able to affect mental health, as well as brain function and plasticity.21 Data supporting the link between nutrition and adult hippocampal neurogenesis are increasingly emerging from studies in animal models. Among others, a recent study demonstrated that intermittent fasting can have a neuroprotective effect through the activation of different signaling pathways, and that a murine model fasted intermittently shows increased hippocampal neurogenesis versus sedentary mice fed an ad libitum diet.22 Rats fed a diet rich of saturated fats and refined sugar experienced insulin resistance and decreased neurogenesis and neuronal plasticity, together with issues in memory and learning.23,24 Finally, a novel study involving a group of preadolescent children revealed a negative association between sugar intake and the performance in Torrance Test of Creative Thinking, while dietary fiber intake was positively associated with results obtained in this creativity standardized test, where the hippocampus is known to be involved.25\n\n\nGut microbiota and mental health: a well-established relationship\n\nThe microbiota, can be defined as the totality of bacteria, archaea, fungi, viruses, and protozoa that inhabits an organism as commensal microorganisms, and represents a unique fingerprint that characterize every subject, with a marked variability, depending on geographical area, diet, and use of antibiotics and other drugs.26 The gut microbiota, a biomass of up to 2 kg in adult humans, has a vital role in human health: it is able to break down dietary fibers and other food components that we cannot digest, produces vitamins, stimulates the immune system maturation, and prevents the gut colonization by pathogenic species.27 Among others, gut microbiota can produce or stimulate the release of several substances that can reach the brain through the circulation and cross the blood–brain barrier (BBB). Evidence has highlighted a key role for microbiota as a link between the gut and essential brain processes, such as microglial activation, neurogenesis and myelination, involved in cognition, mood and behavior, as recently reviewed elsewhere.28 Gut microbes can influence T and B lymphocytes in the wall of the gut, with consequences on the release of pro-inflammatory cytokines that in turn can increase the BBB permeability.29 The vagus nerve represents another route from the gut to the brain.27\n\nAn approach based on the experimental manipulation of gut microbiota across natal and adult environments is leading to a better comprehension of the interplay between cognitive variation and the gut microbial community. In this setting, studies in murine models showed that the maternal gut microbiome can affect the neurodevelopment during key prenatal periods, without any environmental challenge, through the release in maternal serum of microbiota-dependent metabolites.30 Faecal microbiota transplant (FMT) from donor mice of different age to young adult mice revealed the detrimental effect of FMT from aged donors on central nervous system, thus supporting the relevance of the gut-brain axis in ageing and providing a basis to test therapies targeting the microbiota in elderly subjects to improve their cognitive functions.31 A fine-tuning of the brain-gut axis has been highlighted also in humans by the recent findings of a link between brain behavior and development in the offspring and the composition of maternal prenatal gut microbiota.32 Moreover, patients with an imbalance in function and composition,27 i.e. a range of psychiatric and neurological disorders exhibit versus healthy controls of gut microbiota.33 In turn, diseases, as well as disease-associated factors, such as drug intake or a very different diet, can influence the composition of gut microbiota.27\n\nA high-quality diet may be of support in the modulation of gut microbiota at short- and long-term, reduce brain inflammation and stress and subsequently preserve a good cognitive function. Dietary factors can directly shape the gut microbiota composition in both rodents and humans.6 A meta-analysis including gut microbiota studies highlighted the unique effects of the Mediterranean diet on intestinal microbiota.34 However, dietary patterns may affect not only the gut microbiota but also the intestinal permeability, and in turn affect the mental health. In more detail, an increased permeability induced, among others, by a high-fat diet, may allow the activation of the immune cells in the intestinal wall by the lipopolysaccharides produced by bacteria, finally leading to a higher systemic inflammation.35\n\nIn addition to a high-quality diet, the use of prebiotics and probiotics and of specific food supplements (e.g. alpha-tocopherol) could be helpful to modify the gut microbiota and restore a healthy status.26,36 In the context of mental health, some placebo-controlled studies in humans have reported effects of probiotics on emotion- and stress-related parameters. Among others, a double-blind, randomized, placebo-controlled clinical trial on patients with a diagnosis of major depressive disorder showed that the administration of probiotic supplements for 8 weeks led to an improvement of mood, in addition to a decrease of insulin levels and of high-sensitivity C-reactive protein in the serum.37 More recently, the administration for 24 weeks of tablets containing heat-inactivated Lactobacillus gasseri CP2305 to healthy subjects under stressful conditions led to an improvement of sleep quality, mental state, and gut microbiota.38 A larger number of studies and of greater size is required to further support a direct effect of probiotics on brain function, possibly with greater standardization of strains, doses, and measure of study outcomes.27 Finally, a special class of probiotics, the psychobiotics, has been recently defined based on their ability to produce or stimulate the production of neurotransmitters, enteroendocrine hormones and other molecules, with a large range of applications, including mood and stress alleviation.39 A beneficial role for probiotics in mitigating anti-depressive effects, both as an adjunct to antidepressant drugs or as a stand-alone intervention, has been recently suggested elsewhere.40 This is of course an interesting field with huge potentialities; however, the administration of psychobiotics in the general population should be carefully evaluated and supported by more evidence.\n\n\nThe burden of mental health disorders in general population\n\nSeveral disorders, such as anxiety, stress, sleep disturbance, can affect both the mental well-being and the physical health. Stress can be defined as a reaction of the body to the impairment of a condition of equilibrium and is a common problem in several societies, where there are different types of pressures.41 Stress can prolong the release by adrenal glands of the steroid hormone cortisol and to subsequent effects on immune cells and on the production of free radicals.42 When in excess, the free radicals reduce the antioxidant stock in our body, leading to a damage of molecular and cellular components, and leading to oxidative imbalance and inflammation.43 Stress can contribute to a reduction of the quality of life (QoL) and lead to a final state of exhaustion, possibly implying cardiovascular diseases, skin problems, hypertension, infertility, inflammatory bowel syndrome and diabetes mellitus, among others.41 Unhealthy eating habits may result in an increased level in stress; in more detail, improper nutritional balance, insufficient vitamin intake, and excess consumption can exacerbate the stress response and create an imbalance of stress hormones.41 The Mediterranean diet, based on higher intake of fruit, vegetables, nuts, whole grain, seeds, beans, and higher levels of fibers, and on lower levels of red meat, is protective against inflammation and stress. Stress increases the metabolism and creates a greater physiological demand that can be supported through a supplementation with minerals and vitamins. Stress protection is also conferred by omega-3 fatty acids.41\n\nSleep disorders, such as obstructive sleep apnea and insomnia, can cause an impairment of the circadian rhythm. They can be particularly frequent and relevant in aged subjects, where sleep quality, particularly self-reported sleep quality, and QoL are strictly correlated.44 Sleep has a restorative effect on the endocrine and the immune system, supports the recovery of the nervous system and has a fundamental role in memory, learning and in synaptic plasticity.45 Adequate sleep is fundamental for the preservation of metabolic equilibrium. Disturbances and short duration of the sleep are risk factors for inflammation, that is associated with a higher risk of cardiovascular diseases, such as hypertension,46 metabolic disorders, overweight and obesity.47 Of interest, a recent cross-sectional observational study of 13-15 years old subjects revealed that sleep duration and timing seem to be independently associated with weight status in adolescence, and consequently may be important targets to prevent obesity.48 The HPA may be an important mediator of the effects of sleep disorders on general health and well-being. Cortisol is the main hormone of the HPA axis, and is characterized by circadian rhythmicity.47 Pineal melatonin affects the regulation of the HPA axis mentioned above and displays a dynamic circadian rhythm as well, as it is produced at night from 5-hydroxytryptophan and its production is blocked by intense light.49 A positive association between adequate sleep and a health-related lifestyle, including the choice of a healthy dietary pattern, has been shown in subjects of different age. Different studies showed that subjects who sleep less tend to prefer high-energy foods, with a lower intake of vegetables. But ingested food can affect sleep quantity and quality as well. In particular, foods affecting the availability of the essential amino acid tryptophan, as well as the biosynthesis of melatonin and serotonin, may be the most beneficial in sleep induction.50\n\nAnxiety can be defined as an emotion characterized by worried thoughts, tension, and physical symptoms, such as rapid heartbeat, higher blood pressure, sweating, dizziness, in the absence of an immediate threat. Anxiety can occur occasionally in healthy individuals, but it becomes pathological if disproportionate and persistent. The estimated global prevalence rate of anxiety disorders is currently about 28%.51 Anxiety and depressive disorders are associated with increased inflammatory activity, HPA-axis hyperactivity, and an increased tone of the autonomic nervous system.52 Knowledge about the main roles of bioelements in anxiety disorders increased in the latest years.53 In more detail, deficiency of some elements can lead to an excessive release of glutamate and to excessive activation of the HPA axis. Conversely, the regulatory function of bioelements in the body can possibly lead to anxiolytic-like effects, by virtue of their ability to modulate neurotransmission, as well reviewed elsewhere.54 Of interest, a cross-sectional study has recently highlighted that increased intake of micronutrients acting as methyl donors was related to a reduced probabilities of psychological disorders, as better described below.55 These data were confirmed by another cross-sectional study, showing an inverse relationship between anxiety and dietary intake of B vitamins, with the dietary intake of biotin associated with a lower risk of stress and anxiety only in female subjects.56\n\nLow cognitive performance, including low concentration, learning, memory and reasoning, may be due to fatigue, tiredness, stress. An optimal cognitive performance is fundamental during all stages of life. During childhood, the cognitive performance is critical to optimize brain development, while during adulthood it is important to maintain a good cognitive functioning. Finally, during aging, it is of primary importance to delay the cognitive decline, considered as a phase of transition between healthy aging and dementia.57 A healthy diet, rich in PUFAs, polyphenols and other nutritional supplements, such as vitamins, can be beneficial for cognitive performance.6\n\nThe global prevalence of common mental health disorders among adults has been examined fundamentally by two studies: the first one, published by Steel et al. in 2014, estimated a lifetime prevalence of 9.6% for mood disorders, 29.1% for all mental disorders, 12.9% for anxiety, and 3.4% for substance abuse.58 The second study estimated a global prevalence of anxiety and depression of 3.6% and 4.4% respectively, with a greater prevalence in female subjects versus male subjects (https://apps.who.int/iris/bitstream/handle/10665/254610/WHO-MSD-MER-2017.2-eng.pdf).\n\nThe global prevalence of the mental health disorders described above in the general population has shown a dramatic increase with the recent COVID-19 pandemic, as highlighted by a systematic review and meta-analysis including participants from about 30 Countries. In more detail, the global prevalence estimate following the COVID-19 outbreak was 26.9% for anxiety, 36.5% for stress, 28.0% for depression, 50.0% for psychological distress, 24.1% for post-traumatic stress symptoms and 27.6% for sleep disorders.59\n\n\nThe omega-3 fatty acids role in mental health and well-being\n\nDietary fats or lipids are not only a main source of energy, but play a role also in several other processes, e.g. as components of cell membranes, regulators of enzymatic activity, precursors of bioactive molecules, regulators of gene expression. Fatty acids are classified on the basis of the number of double bonds in their molecules; among them, PUFAs have ≥2 double bonds, mostly in the cis configuration. PUFAs consist of 3 fatty acids: DHA, alpha- linoleic acid (ALA) and EPA. The human body can synthetize the n-3 PUFAs EPA and DHA from ALA through a series of desaturation, elongation and β-oxidation reactions occurring first in the endoplasmatic reticulum and then in the peroxisomes of animal tissues. ALA is considered an essential fatty acid (EFA) as it is not synthetized by the body.60 ALA is found in some vegetables, while DHA and EPA are abundant in fish, cultivated marine algae and human milk. Foods enriched with n-3 PUFAs and supplements are also available as DHA and EPA source.60\n\nAbout 20% of the dry weight of the brain and 1/3 of all fats in the central nervous system are composed by omega-3 PUFAs; they maintain membrane fluidity for synaptic signaling process, regulate neuronal membrane excitability, improve memory and learning.61\n\nThe adequate daily intake for DHA and EPA for adults is 250-500 mg, based on cardiovascular risk considerations.60\n\nOmega-3 PUFAs EPA and DHA have both independent and shared effects on the brain, and represent a potential treatment for several neurodegenerative and neurological disorders by virtue of their neuroprotective properties.62 DHA is the most abundant n-3 PUFA in the brain and is part of the structural lipids in cell membranes, in particular of phospholipids in the retina and in the nervous tissue, with a major role in maintenance of neuronal membranes, in maintenance of normal brain functions and in cognitive process (Figure 1).62 DHA and EPA can be converted in their respective endocannabinoid derivatives, docosahexaenoyl-ethanolamine and eicosapentaenoyl-ethanolamine, showing possible antidepressive effects.63 In addition, specialized proresolving mediators, such as protectins, resolvins, lipoxins, and maresins, are bioactive lipids derived from omega-3 PUFAs produced by innate immune cells. This class of lipids counteracts oxidative stress and inflammation promoting the resolution of inflammation.64 DHA and EPA blood levels can be affected both by genetic polymorphisms and by gender.62,65\n\n(↓): downregulation; (↑): upregulation.\n\nBased on current literature, it has been established that DHA supports a healthy brain development as one of its building blocks and supports the nerve transmission; DHA also may support the working memory, concentration levels and the brain performance in aging adults.66 The developing brain accumulates large amounts of DHA, in particular during the first years of life; it is acquired mainly from the mother through the placental transfer in the pre-natal period and though the breast milk post-natally. However, the brain increases its capacity to synthesize DHA in parallel with gestational age.67 Studies in murine models submitted to psychological stress revealed the important role of a balanced diet in supporting brain development for later cognitive function. In more detail, the studies showed an improvement in cognitive behaviors, memory and plasticity markers in the brain of adolescence in rats fed a diet enriched with the omega-3 PUFAs, EPA, DHA, and vitamin A. Of interest, the protective effect of the PUFA-enriched diet were preserved during adulthood, long after the termination of the exposure to a stressful environment.68 A very recent study confirmed the positive role of dietary DHA on the preservation of cognitive function also in aged rats fed a processed foods diet.69\n\nMoving to data derived from observational studies and trials in humans, an inverse correlation between plasma levels of EPA, DHA and total omega-3 PUFAs and cognitive impairment, dementia risk and depressive symptoms in the elderly has been highlighted in several studies.19,62,70–72 It has also been suggested that an adequate dietary intake of omega-3 PUFAs can slow the age-related mild cognitive impairment (MCI) and protect against the risk of dementia.73 Literature data also indicate that the impact of stress can be limited by an adequate amount of DHA in the brain.73 Matura et al. (2021)74 investigated the relationship in cognitively healthy individuals between cognitive performance, dietary fat composition and brain morphology, showing that a high ratio between saturated fatty acids and (n-3) PUFAs has a significant correlation with lower grey matter volumes and poorer verbal memory performance in areas of the left prefrontal cortex, a region involved in memory processes, thus suggesting that a diet rich in PUFAs could positively affect the morphology in brain areas important for executive functions and memory. A trial on healthy volunteers revealed that the Zone diet and omega-3 PUFAs were able to increase well-being and to modulate the mood state, as revealed by ad-hoc questionnaires. The supplementation with omega-3 PUFAs was related to an increase of vigor and a decrease of negative factors such as angry, anxious and depressive mood in healthy subjects.75 Several other trials supported the effectiveness of a PUFA-enriched diet on the preservation of cognitive functions in the elderly.76\n\nOf interest, some studies indicated that an inter-individual variability in omega-3 PUFA clinical response seems to be related to genetic variants, especially in APOE4 carriers,77 and to epigenetic modifications of genes involved in PUFA metabolism and associated with the risk of mental disorders, such as autism spectrum disorders and attention deficit/hyperactivity disorder,78,79 thus supporting an approach based on subject stratification, as indicated by the emerging “precision nutrition” concept.\n\n\nThe magnesium role in mental health and well-being\n\nMagnesium is an essential ion to the human body, as it is the second most abundant intracellular cation after potassium, with a daily reference intake of 375 mg.80 In more detail, the Recommended Dietary Allowance (RDA) for magnesium is 400–420 mg for male subjects and 310–320 mg for female subjects with age >19 years (https://ods.od.nih.gov/factsheets/Magnesium-HealthProfessional/). Magnesium acts as a physiological calcium antagonist, and is involved in hundreds of enzymatic reactions, including those related to protein synthesis and energy metabolism, DNA stability and DNA repair as well as the preservation of the electrical potential of nerve tissue and cell membranes. Magnesium contributes to normal electrolyte balance and to the normal function of the nervous system, as well as to the reduction of fatigue and tiredness, particularly in situations of inadequate micronutrient status, and its reserve tends to be exhausted by our body in times of increased stress, so it is fundamental to ensure a proper intake of this mineral (Figure 1).81\n\nReduced magnesium levels have been associated to migraine, depression, epilepsy. It regulates N-Methyl-D-aspartate (NMDA) excitability, so that in magnesium deficiency NMDA receptors become hyperexcitable and this condition can be further amplified by the inhibitory action of gamma-aminobutyric acid (GABA) receptors, which are regulated by magnesium. Several authors hypothesized that magnesium may relieve depression by blocking NMDA receptors that are involved in depression development.82 Moreover, it has been shown that magnesium plays anti-inflammatory and anti-depressant effects.83,84 Interestingly, a magnesium uptake leads to a decrease in body weight and a loss of fat mass which is accompanied by a decrease in serum inflammatory parameters. There is a bidirectional relationship between obesity and depression as the first is characterized by a chronic inflammation of low-grade, with involvement of the stress axis. Obesity can increase by 55% the risk of comorbid depression, that in turn leads to a 58% increased risk of obesity.85 Clinical studies have shown that hypomagnesemia correlates with the severity of depressive symptoms86 and with the onset of metabolic syndrome and diabetes often associated with obesity conditions. Magnesium deficiency affects energy metabolism and may stimulate the onset of inflammation-associated metabolic disorders, particularly in obese patients. In addition, hypomagnesemia may increase oxidative stress by acting on the activities of antioxidant enzymes such as glutathione, catalase and superoxide dismutase. Magnesium was shown to enhance synaptic plasticity by inducing increased levels of presynaptic synapsin 1, PSD93 and PSD95. Magnesium exerts an anti-inflammatory activity through the regulation of the NF-kB/Nrf2 signaling pathways.87 Magnesium supplementation was able to improve memory and learning in patients affected by dementia and in healthy animals and was shown to play a key role in neuronal maturation.88\n\nThe relationship between magnesium concentration in the serum and mental health has been highlighted in a cross-sectional study that showed an association between low magnesium concentration in the serum and greater perceived stress in healthy women without psychiatric disorders.89 Consistently, magnesium supplementation has been shown to be beneficial in stress alleviation in a phase IV, investigator-blinded trial enrolling healthy subjects with severe/extremely severe stress and low magnesemia and randomized 1:1 to magnesium–vitamin B6 combination or magnesium alone and treated accordingly for 8 weeks.90 Other studies showed a positive effect of magnesium supplementation on biomarkers and symptoms of stress and on sleep disorders. In a double-blind, randomized trial on healthy elderly subjects, magnesium supplementation improved subjective measures of insomnia, and, likewise, insomnia objective measures, such as concentration of serum cortisol, renin and melatonin.91 A very recent systematic review and meta-analysis of 3 randomized control trials comparing oral magnesium to placebo in older adults in 3 different Countries did not led to statistically significant data, but however it supported the use of oral magnesium supplements for insomnia symptoms.92 The authors suggested the need for additional well-designed clinical trials due to the substandard quality of currently available studies.\n\n\nThe role of alpha-tocopherol in mental health and well-being\n\nPlant-derived alpha-tocopherol is one of the eight isoforms of vitamin E. It increases the regularity of lipid packaging in cell membranes and is part of the antioxidant defense system, functioning physiologically as a chain-breaking antioxidant that protects polyunsaturated fatty acids from peroxidation (Figure 1).93 The reference alpha-tocopherol daily dietary intake for adult subjects is 12 mg.94 Major dietary vitamin E sources, are fortified ready-to-eat cereals; greens such as spinach; nuts, seeds; and vegetable oils, in particular safflower and sunflower.94 Vitamin E can be stored in the fatty tissue, and is located mainly within the cell membranes, where it protects against lipid peroxidation, a consequence of oxidative stress, counteracting reactive oxygen species formation. Alpha-tocopherol is involved in the inhibition of free radicals production, whereas gamma-tocopherol neutralizes the free radicals already present.95\n\nStudies focusing on alpha-tocopherol deficiency involving humans or animal models established the critical role of this molecule in protecting the central nervous system, and in particular the cerebellum, from motor coordination deficits and oxidative damage. Emerging data also demonstrate the critical roles of alpha-tocopherol in preserving memory, emotive responses and learning, as reviewed elsewhere.96\n\nSeveral studies in animal models supported a positive role for alpha-tocopherol in preservation of cognitive function and sleep quality. Among others, a preclinical study in a murine model showed the neuroprotective effect of vitamin E on memory impairment induced by chronic sleep deprivation.97 In addition, a chronic treatment of old rats with alpha-tocopherol significantly increased in a dose- and/or time-dependent manner the synthesis rate and the levels of monoaminergic neurotransmitters (noradrenaline, dopamine, serotonin) in striatum and in hippocampus, both involved in motor coordination and memory processing.98 Moving to humans, a study involving 124 young male subjects showed a significant association between self-reported poor sleep quality and low intakes of some nutrients, including alpha-tocopherol.99 These findings are in agreement with the association between sleep regulation and oxidative stress suggested by previous reports,100 based on the hypothesis that the antioxidant properties of alpha-tocopherol could improve the quality of the sleep. However, additional investigations are necessary to explore the potential beneficial effect of alpha-tocopherol on sleep quality. A meta-analysis evaluating the association between intakes of the most common 3 antioxidants (β-carotene, vitamin C, vitamin E) and the risk of Alzheimer’s disease revealed that the intake of the 3 antioxidants is able to reduce the risk of Alzheimer’s disease, and that vitamin E exhibits the most relevant protective effect.101 In addition, serum alpha-tocopherol resulted to be significantly correlated with cognitive function in a population of older subjects.102 However, these findings have not been confirmed by other studies,103 including the Ginkgo Evaluation of Memory observational study, based on a large older population, where plasma antioxidants, including alpha-tocopherol, were not significantly related to future risk of dementia, Alzheimer’s disease or cognitive decline before dementia diagnosis.104\n\nThe synergistic effect of vitamin E administered together with omega-3 PUFAs has been related to the complementation of the anti-oxidant and anti-inflammatory activity exhibited by the two different classes of compounds. It is well known that oxidative stress is one of the main inducers of neural cell damage and death in neurodegenerative diseases.105\n\nThe CARES Trial 1 evaluated the role of a specific nutritional protocol to improve the cognitive performance in subjects affected by MCI. Preliminary results were encouraging and showed improved performance in episodic memory and global cognitions in MCI patients, who have been following a diet enriched with omega-3 PUFAs and vitamin E for 12 months.106\n\nInterestingly, a recent study suggested that the levels of circulating alpha-tocopherol do not provide a complete insight in vitamin E antioxidant capacity and activity. Special attention in observational and efficacy studies should be devoted not only to alpha-tocopherol plasma concentration, but also to its metabolism, marked by α-carboxymethyl-hydroxychroman and α-tocopheronolactone hydroquinone levels in the urine, as potential reflection of lipid oxidation.107 Moreover, discrepancies among study results could be due to confounding factors or to different methods for antioxidant measurement in the body. In light of these considerations, additional trials are needed, with a special focus on redox potential of alpha-tocopherol in the setting of cognitive impairment and on preclinical phases of disease, with the enrollment in studies of younger populations.\n\n\nThe folic acid role in mental health and well-being\n\nVitamin B9, also known as folate, is a water-soluble vitamin, abundant in the liver and in vegetables and whose chemical structure include a pteridine ring, p-aminobenzoic acid, and gamma-linked glutamate residues. Daily reference intake for folate is 200 μg. Folic acid is the oxidized monoglutamate synthetic form of folate, quite rare in nature.108 Both folic acid and folate must be metabolized to the metabolically active form L-methylfolate through a multi-steps process to play their roles. L-methylfolate can cross the BBB and regulate the production of the neurotransmitters serotonin, norepinephrine and dopamine, thus contributing to mental function and performance.109 Folic acid is essential for the production and maintenance of new cells, RNA and DNA nucleotide synthesis, and consequently for cell division. Folic acid is also essential for the remethylation of homocysteine, acting as a coenzyme for one-carbon metabolism (Figure 1).108 Folate plays a key role in the methionine cycle, which is involved both in the conversion of homocysteine to methionine and in the production of S-adenosylmethionine, a universal donor of the methyl group.110 Elevated homocysteine levels have been linked to atrophy of the cerebral cortex and loss of magnesium: both of these effects can be reversed by vitamins B.111–113\n\nFolic acid is necessary for mental function and performance, playing a role in learning, memory and reasoning, concentration, and resistance to stress,114 and its deficiency is linked to the development of forgetfulness in mild form and irritability.110 A relevant contribution to the comprehension of the role of folic acid during development came from the examination of the effects of folic acid deficient or replete diets in mice, containing either normal folic acid intake or no source of folate.115 Folic acid deficient mice exhibited a significant impairment of memory versus control mice, associated with significant gene expression changes specific to the hippocampus. Folic acid supplementation alleviated the cognitive decline related to age and inhibited the apoptosis of neurocytes in a murine model of accelerated senescence, and the mechanism potentially involved could be the alleviation of telomere attrition,116 since telomere length shortening has been related to MCI and Alzheimer’s disease development.117 However, data on the effect of folic acid supplementation on cognitive performance in humans are contrasting. Among them, Bryan et al. investigated the effects of a supplementation for 35 days with folate, vitamin B12, vitamin B6 or with placebo in healthy women included in a wide age range.118 In addition, they examined the association between dietary intake of these vitamins and mood and cognition. Supplementation induced a positive effect on some measures of memory performance, but not on mood. Dietary intake status was associated with verbal ability, recall and recognition and speed of processing.118 Conversely, a randomized controlled trial enrolling elderly people with elevated homocysteine levels treated for two years with vitamin B12 and folic acid did not experience any beneficial effect on cognitive performance.119 Similar results have been obtained in another randomized clinical trial enrolling older MCI patients with high levels of homocysteine and supplemented for two years with methylcobalamin and folic acid. However, in this study the supplement led to a significant, even if not sustained, reduction in depressive symptoms at month 12.120 Of interest, a cross-sectional study showed that a moderate folic acid intake was associated with lower ratios of depressions in a general population.56 Finally, dietary methyl donor micronutrients intake (choline, folate, betaine, B6 and B12 vitamins methionine) was linked to reduced odds of psychological disorders, in particular in women and obese or overweight subjects.55\n\n\nFood supplements for mental health and well-being\n\nAs reported above, stress is a common problem and induces greater physiological demands: more oxygen, circulation, energy, and consequently more metabolic cofactors are necessary.1,41 Among others, the psychosocial stress experienced by subjects is particularly difficult to prevent. Nutritional interventions could confer resilience and rescue stress vulnerability, as reviewed above and elsewhere.121 Moreover, according to recent clinical observations, specific nutrients could be helpful also in complex neuropsychiatric conditions, such as major depressive syndrome, bipolar disorders, and obsessive-compulsive disorder, among others.122\n\nThe dietary patterns leading to positive and protective effects against stress and inflammation are consistent with the Mediterranean diet characteristics described above.9,123 Stress reduction is also supported by supplementation of minerals and vitamins. Additional stress protection is provided by the intake of omega-3 PUFAs.41\n\nDeficiencies of micro- and macronutrients can be due to, among others, insufficient intake, concomitant diseases, aging, drugs, lifestyle, metabolic disorders and genetic alterations, and can lead to different outcomes. The relative lack of omega-3 PUFAs in the Western diet can lead to insufficient DHA in brain cell membranes. An insufficient omega-3 PUFA dietary supply may be particularly relevant during aging as studies in murine models revealed that the aging brain tend to lose DHA. The specific decrease of DHA in aged brain may be due to an age-associated reduction of some enzymatic activities, specifically involved in the incorporation of DHA into brain phospholipids. This process can be prevented through dietary supplementation of DHA, as shown in aged rats.73\n\nFor what concerns magnesium, hypomagnesemia can be genetically determined or be consequent to the use of certain types of drugs or to alcohol abuse, or can follow gastrointestinal loss or dysregulation of renal reabsorption in critically ill patients.82 Magnesium deficiency always includes secondary electrolyte disturbances.124 Alpha-tocopherol deficiency because of inadequate intake of vitamin E is not common and can occur more frequently in children than in adults, in the setting of chronic fat malabsorption or because of genetic abnormalities in hepatic alpha-tocopherol transfer protein or in lipoprotein synthesis. The development of clinical evidence of vitamin E deficiency can take several years of exposure to extremely low vitamin E levels and can impair the immune function, increase adverse events during pregnancy and distorts the carefully programmed development of the nervous system, leading to defects also in several developing organs.125 Low folate intake or an anomalous folate metabolism as well as a deficiency of vitamin B12 or B6 induce homocysteine elevation, and hyperhomocysteinemia has been linked to a higher risk of cerebrovascular and cardiovascular disorders.126 In addition, folate deficiency impairs DNA replication and cell division, with negative effects in particular on proliferating tissues, such as bone marrow.110 Specifically-designed food supplements could represent a useful tool to limit the impact of disorders such as anxiety, stress, sleep disturbance, and help to preserve the cognitive function throughout life without the side effects of drugs. Currently marketed food supplements whose ingredients include the micro- and macronutrients folic acid, magnesium, PUFAs and alpha-tocopherol, are listed in Table 1. Among them, Libretto® appears of particular interest, as a single capsule fulfills the 100% of the recommended daily reference intake of folic acid according to Reg. (EU) n. 1169/2011, the 29% of magnesium and the 21% of vitamin E recommended daily reference intake, respectively (Table 2). Food supplements specific for memory loss, Alzheimer’s disease and MCI have been marketed as well in the latest years as adjunct of therapies to compensate possible nutrient deficiencies, and their efficacy has been evaluated in several meta-analyses, with different results.122\n\n* NRV daily reference intakes for vitamins and minerals (adults) according to Reg. (EU) n° 1169/2011.\n\n\nConclusions\n\nThe micro- and macronutrients folic acid, magnesium, alpha-tocopherol and the omega-3 PUFAs EPA and DHA play individual and shared neuroprotective effects in mental health and brain aging. Unfortunately, existing studies to inform dietary recommendations for patients with mood, anxiety or sleep disorders are often limited by study size, heterogeneous patients’ populations, and poor methodology. Well-designed observational studies or clinical trials will be of major relevance in the field of diet and dietary supplements for mental health and well-being.\n\nThe future development in the field of mental health and nutrition will probably combine psychological and psychiatric research with neuroscience and nutritional genomics. The growing application of the emerging concept of “precision nutrition”, i.e. a subject stratification based not only on genetics but also on other variables, such as the profiling of microbiota, physical activity, smoking habits, concurrent medications and other features, will probably increase the ability of dietary interventions to improve mental health and well-being. Considering the increasing burden of mental and neurological disorders and considering that food is a universally modifiable risk factor, even small improvements in nutritional habits could lead to large improvements in mental health and well-being in the global population, while improving the sustainability of healthcare systems by reducing the costs associated with cognitive decline and poor mental health. In addition to increased awareness of the importance of nutrition and to consequent improvement of nutritional habits, the growing development and use of targeted, well-balanced food supplements aiming to support the mental health and well-being will probably represent another relevant tool in future decades.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgments\n\nAmalia Forte, Ph.D., provided the medical writing support on behalf of Menthalia S.r.l., Naples, Italy.\n\n\nReferences\n\nBusinaro R, Ippoliti F, Ricci S, et al.: Alzheimer's disease promotion by obesity: induced mechanisms-molecular links and perspectives. Curr. Gerontol. Geriatr. Res. 2012; 2012: 986823. PubMed Abstract \| Publisher Full Text\n\nBusinaro R, Vauzour D, Sarris J, et al.: Therapeutic Opportunities for Food Supplements in Neurodegenerative Disease and Depression. Front. Nutr. 2021 May 14; 8: 669846. 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PubMed Abstract \| Publisher Full Text\n\nYurko-Mauro K, Alexander DD, Van Elswyk ME: Docosahexaenoic acid and adult memory: a systematic review and meta-analysis. PLoS One. 2015 Mar 18; 10(3): e0120391. PubMed Abstract \| Publisher Full Text\n\nPatrick RP: Role of phosphatidylcholine-DHA in preventing APOE4-associated Alzheimer's disease. FASEB J. 2019 Feb; 33(2): 1554–1564. PubMed Abstract \| Publisher Full Text\n\nSun C, Zou M, Wang X, et al.: FADS1-FADS2 and ELOVL2 gene polymorphisms in susceptibility to autism spectrum disorders in Chinese children. BMC Psychiatry. 2018 Sep 4; 18(1): 283. Publisher Full Text\n\nBrookes KJ, Chen W, Xu X, et al.: Association of fatty acid desaturase genes with attention-deficit/hyperactivity disorder. Biol. Psychiatry. 2006 Nov 15; 60(10): 1053–1061. PubMed Abstract \| Publisher Full Text\n\nEFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA): Scientific Opinion on the substantiation of health claims related to magnesium and “hormonal health” (ID 243), reduction of tiredness and fatigue (ID 244), contribution to normal psychological functions (ID 245, 246), maintenance of normal blood glucose concentrations (ID 342), maintenance of normal blood pressure (ID 344, 366, 379), protection of DNA, proteins and lipids from oxidative damage (ID 351), maintenance of the normal function of the immune system (ID 352), maintenance of normal blood pressure during pregnancy (ID 367), resistance to mental stress (ID 375, 381). EFSA J. 2010; 8(10): 1807. Publisher Full Text\n\nKirkland AE, Sarlo GL, Holton KF: The Role of Magnesium in Neurological Disorders. Nutrients. 2018 Jun 6; 10(6): 730. PubMed Abstract \| Publisher Full Text\n\nde Baaij JH , Hoenderop JG, Bindels RJ: Magnesium in man: implications for health and disease. Physiol. Rev. 2015 Jan; 95(1): 1–46. Publisher Full Text\n\nAbiri B, Sarbakhsh P, Vafa M: Randomized study of the effects of vitamin D and/or magnesium supplementation on mood, serum levels of BDNF, inflammation, and SIRT1 in obese women with mild to moderate depressive symptoms. Nutr. Neurosci. 2021 Jul 2; 1–13. Publisher Full Text\n\nAbiri B, Vafa M: Effects of vitamin D and/or magnesium supplementation on mood, serum levels of BDNF, inflammatory biomarkers, and SIRT1 in obese women: a study protocol for a double-blind, randomized, placebo-controlled trial. Trials. 2020 Feb 26; 21(1): 225. PubMed Abstract \| Publisher Full Text\n\nKöhler O, Benros ME, Nordentoft M, et al.: Effect of anti-inflammatory treatment on depression, depressive symptoms, and adverse effects: a systematic review and meta-analysis of randomized clinical trials. JAMA Psychiatry. 2014 Dec 1; 71(12): 1381–1391. Publisher Full Text\n\nWidmer J, Henrotte JG, Raffin Y, et al.: Relationship between erythrocyte magnesium, plasma electrolytes and cortisol, and intensity of symptoms in major depressed patients. J. Affect. Disord. 1995 Jun 8; 34(3): 201–209. PubMed Abstract \| Publisher Full Text\n\nOrhan C, Tuzcu M, Deeh Defo PB, et al.: Effects of a Novel Magnesium Complex on Metabolic and Cognitive Functions and the Expression of Synapse-Associated Proteins in Rats Fed a High-Fat Diet. Biol. Trace Elem. Res. 2021 Feb 16; 200: 247–260. PubMed Abstract \| Publisher Full Text\n\nYamanaka R, Shindo Y, Oka K: Magnesium Is a Key Player in Neuronal Maturation and Neuropathology. Int. J. Mol. Sci. 2019 Jul 12; 20(14): 3439. PubMed Abstract \| Publisher Full Text\n\nJung KI, Ock SM, Chung JH, et al.: Associations of serum Ca and Mg levels with mental health in adult women without psychiatric disorders. Biol. Trace Elem. Res. 2010 Feb; 133(2): 153–161. PubMed Abstract \| Publisher Full Text\n\nPouteau E, Kabir-Ahmadi M, Noah L, et al.: Superiority of magnesium and vitamin B6 over magnesium alone on severe stress in healthy adults with low magnesemia: A randomized, single-blind clinical trial. PLoS One. 2018 Dec 18; 13(12): e0208454. PubMed Abstract \| Publisher Full Text\n\nAbbasi B, Kimiagar M, Sadeghniiat K, et al.: The effect of magnesium supplementation on primary insomnia in elderly: A double-blind placebo-controlled clinical trial. J. Res. Med. Sci. 2012 Dec; 17(12): 1161–1169. PubMed Abstract\n\nMah J, Pitre T: Oral magnesium supplementation for insomnia in older adults: a Systematic Review & Meta-Analysis. BMC Complement Med. Ther. 2021 Apr 17; 21(1): 125. PubMed Abstract \| Publisher Full Text\n\nEFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA): Scientific Opinion on the substantiation of health claims related to vitamin E and protection of DNA, proteins and lipids from oxidative damage (ID 160, 162, 1947), maintenance of the normal function of the immune system (ID 161, 163), maintenance of normal bone (ID 164), maintenance of normal teeth (ID 164), maintenance of normal hair (ID 164), maintenance of normal skin (ID 164), maintenance of normal nails (ID 164), maintenance of normal cardiac function (ID 166), maintenance of normal vision by protection of the lens of the eye (ID 167). EFSA J. 2010; 8(10): 1816. Publisher Full Text\n\nTraber MG, Manor D: Vitamin E. Adv. Nutr. 2012 May 1; 3(3): 330–331, 331. PubMed Abstract \| Publisher Full Text\n\nMitra S, Natarajan R, Ziedonis D, et al.: Antioxidant and anti-inflammatory nutrient status, supplementation, and mechanisms in patients with schizophrenia. Prog. Neuro-Psychopharmacol. Biol. Psychiatry. 2017 Aug 1; 78: 1–11. PubMed Abstract \| Publisher Full Text\n\nUlatowski LM, Manor D: Vitamin E and neurodegeneration. Neurobiol. Dis. 2015 Dec; 84: 78–83. Publisher Full Text\n\nAlzoubi KH, Khabour OF, Rashid BA, et al.: The neuroprotective effect of vitamin E on chronic sleep deprivation-induced memory impairment: the role of oxidative stress. Behav. Brain Res. 2012 Jan 1; 226(1): 205–210. PubMed Abstract \| Publisher Full Text\n\nRamis MR, Sarubbo F, Terrasa JL, et al.: Chronic α-Tocopherol Increases Central Monoamines Synthesis and Improves Cognitive and Motor Abilities in Old Rats. Rejuvenation Res. 2016 Apr; 19(2): 159–171. PubMed Abstract \| Publisher Full Text\n\nMatsunaga T, Nishikawa K, Adachi T, et al.: Associations between dietary consumption and sleep quality in young Japanese males. Sleep Breath. 2021 Mar; 25(1): 199–206. PubMed Abstract \| Publisher Full Text\n\nTrivedi MS, Holger D, Bui AT, et al.: Short-term sleep deprivation leads to decreased systemic redox metabolites and altered epigenetic status. PLoS One. 2017 Jul 24; 12(7): e0181978. PubMed Abstract \| Publisher Full Text\n\nLi FJ, Shen L, Ji HF: Dietary intakes of vitamin E, vitamin C, and β-carotene and risk of Alzheimer's disease: a meta-analysis. J. Alzheimers Dis. 2012; 31(2): 253–258. PubMed Abstract \| Publisher Full Text\n\nNiemchick KL, Riemersma C, Lasker GA: Lipophilic Antioxidants and Cognitive Function in the Elderly. Nutr Metab Insights. 2020 Feb 3; 13: 1178638820903300. PubMed Abstract \| Publisher Full Text\n\nBrewer GJ: Why vitamin E therapy fails for treatment of Alzheimer's disease. J. Alzheimers Dis. 2010; 19(1): 27–30. PubMed Abstract \| Publisher Full Text\n\nKoch M, Furtado JD, Cronjé HT, et al.: Plasma antioxidants and risk of dementia in older adults. Alzheimers Dement (N Y). 2021 Sep 5; 7(1): e12208. PubMed Abstract \| Publisher Full Text\n\nZakharova IO, Sokolova TV, Vlasova YA, et al.: α-Tocopherol at Nanomolar Concentration Protects Cortical Neurons against Oxidative Stress. Int. J. Mol. Sci. 2017 Jan 21; 18(1): 216. PubMed Abstract \| Publisher Full Text\n\nPower R, Nolan JM, Prado-Cabrero A, et al.: Targeted Nutritional Intervention for Patients with Mild Cognitive Impairment: The Cognitive impAiRmEnt Study (CARES) Trial 1. J. Pers. Med. 2020 May 25; 10(2): 43. PubMed Abstract \| Publisher Full Text\n\nMartens LG, Luo J, Meulmeester FL, et al.: Associations between Lifestyle Factors and Vitamin E Metabolites in the General Population. Antioxidants (Basel). 2020 Dec 15; 9(12): 1280. PubMed Abstract \| Publisher Full Text\n\nEbara S: Nutritional role of folate. Congenit Anom (Kyoto). 2017 Sep; 57(5): 138–141. Publisher Full Text\n\nLeahy LG, Vitamin B: Supplementation: What's the Right Choice for Your Patients?. J. Psychosoc. Nurs. Ment. Health Serv. 2017 Jul 1; 55(7): 7–11. PubMed Abstract \| Publisher Full Text\n\nEFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA): Scientific Opinion on Dietary Reference Values for folate. EFSA J. 2014; 12(11): 3893. Publisher Full Text\n\nDouaud G, Refsum H, de Jager CA , et al.: Preventing Alzheimer's disease-related gray matter atrophy by B-vitamin treatment. Proc. Natl. Acad. Sci. U. S. A. 2013 Jun 4; 110(23): 9523–9528. PubMed Abstract \| Publisher Full Text\n\nJernerén F, Elshorbagy AK, Oulhaj A, et al.: Brain atrophy in cognitively impaired elderly: the importance of long-chain ω-3 fatty acids and B vitamin status in a randomized controlled trial. Am. J. Clin. Nutr. 2015 Jul; 102(1): 215–221. PubMed Abstract \| Publisher Full Text\n\nHama Y, Hamano T, Shirafuji N, et al.: Influences of Folate Supplementation on Homocysteine and Cognition in Patients with Folate Deficiency and Cognitive Impairment. Nutrients. 2020 Oct 14; 12(10): 3138. PubMed Abstract \| Publisher Full Text\n\nEFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA): Scientific Opinion on the substantiation of health claims related to folate and contribution to normal psychological functions (ID 81, 85, 86, 88), maintenance of normal vision (ID 83, 87), reduction of tiredness and fatigue (ID 84), cell division (ID 195, 2881) and contribution to normal amino acid synthesis (ID 195, 2881) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA J. 2010; 8(10): 1760. Publisher Full Text\n\nLawton A, Morgan CR, Schreiner CR, et al.: Folate-Dependent Cognitive Impairment Associated With Specific Gene Networks in the Adult Mouse Hippocampus. Front. Nutr. 2020 Nov 12; 7: 574730. PubMed Abstract \| Publisher Full Text\n\nZhou D, Lv X, Wang Y, et al.: Folic acid alleviates age-related cognitive decline and inhibits apoptosis of neurocytes in senescence-accelerated mouse prone 8: deoxythymidine triphosphate biosynthesis as a potential mechanism. J. Nutr. Biochem. 2021 Jun 6; 97: 108796. PubMed Abstract \| Publisher Full Text\n\nScarabino D, Peconi M, Broggio E, et al.: Relationship between proinflammatory cytokines (Il-1beta, Il-18) and leukocyte telomere length in mild cognitive impairment and Alzheimer's disease. Exp. Gerontol. 2020 Jul 15; 136: 110945. PubMed Abstract \| Publisher Full Text\n\nBryan J, Calvaresi E, Hughes D: Short-term folate, vitamin B-12 or vitamin B-6 supplementation slightly affects memory performance but not mood in women of various ages. J. Nutr. 2002 Jun; 132(6): 1345–1356. Publisher Full Text\n\nvan der Zwaluw NL , Dhonukshe-Rutten RA, van Wijngaarden JP , et al.: Results of 2-year vitamin B treatment on cognitive performance: secondary data from an RCT. Neurology. 2014 Dec 2; 83(23): 2158–2166. PubMed Abstract \| Publisher Full Text\n\nKwok T, Wu Y, Lee J, et al.: A randomized placebo-controlled trial of using B vitamins to prevent cognitive decline in older mild cognitive impairment patients. Clin. Nutr. 2020 Aug; 39(8): 2399–2405. PubMed Abstract \| Publisher Full Text\n\nToyoda A: Nutritional interventions for promoting stress resilience: Recent progress using psychosocial stress models of rodents. Anim. Sci. J. 2020 Jan; 91(1): e13478. PubMed Abstract \| Publisher Full Text\n\nMuscaritoli M: The Impact of Nutrients on Mental Health and Well-Being: Insights From the Literature. Front. Nutr. 2021 Mar 8; 8: 656290. PubMed Abstract \| Publisher Full Text\n\nBusinaro R, Corsi M, Asprino R, et al.: Modulation of Inflammation as a Way of Delaying Alzheimer's Disease Progression: The Diet's Role. Curr. Alzheimer Res. 2018 Feb 22; 15(4): 363–380. PubMed Abstract \| Publisher Full Text\n\nEFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA): Scientific Opinion on the substantiation of health claims related to magnesium and electrolyte balance (ID 238), energy-yielding metabolism (ID 240, 247, 248), neurotransmission and muscle contraction including heart muscle (ID 241, 242), cell division (ID 365), maintenance of bone (ID 239), maintenance of teeth (ID 239), blood coagulation (ID 357) and protein synthesis (ID 364) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA J. 2009; 7(9): 1216. Publisher Full Text\n\nTraber MG: Vitamin E: necessary nutrient for neural development and cognitive function. Proc. Nutr. Soc. 2021 Apr 26; 80: 319–326. PubMed Abstract \| Publisher Full Text\n\nMcCully KS: Chemical pathology of homocysteine. IV. Excitotoxicity, oxidative stress, endothelial dysfunction, and inflammation. Ann. Clin. Lab. Sci. 2009 Summer; 39(3): 219–232. PubMed Abstract" }	[ { "id": "142179", "date": "11 Jul 2022", "name": "Ned D Heindel", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting, well written and timely article focused on the fundamental role of diet/nutrition on brain function. The importance of improved nutritional habits is also discussed, specifically the beneficial effects of micro- and macronutrients in food and supplements.\n\nSome clarifications on the following would improve the manuscript.\n\nPlease add rationale for the focus only on magnesium, folic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha-tocopherol.\n\nA short discussion on the impact of the gut microbiome on brain metabolome would be interesting.\n\nIs there any evidence that other free fatty acids besides omega 3 influence brain function and well being?\n\nIs magnesium the only ion known to impact cognitive responses?\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/11-140
https://f1000research.com/articles/10-1164/v1	17 Nov 21	{ "type": "Research Article", "title": "Online harassment in Japan: Dissecting the targeting of a female journalist", "authors": [ "Aki Tonami", "Mitsuo Yoshida", "Yukie Sano", "Mitsuo Yoshida", "Yukie Sano" ], "abstract": "Harassment on the Internet, particularly on social media such as Twitter, has reached a level where it can, without exaggeration, be characterised as a real-world societal problem in Japan. However, studies on this phenomenon in the Japanese language environment, especially adopting a victim-centric perspective, are rare. In this paper, we incorporated the concept of online harassment and reviewed existing studies about online harassment from Japan and abroad. We then conducted a detailed case analysis of the “flaming” of a female journalist and those who targeted her on Twitter. Based on our analysis, we observed that there were three layers of users who targeted the journalist: influencers, users who responded to the instigation by influencers, and trolls. Each harassed the journalist, but in a different manner. Given Japan’s particular difficulty of imposing domestic regulations on social media companies that are mostly from abroad, we propose and describe possible measures that individuals and their employers should consider taking.\nソーシャルメディア上の「炎上」はもはや社会問題といっても過言ではないが、日本語環境の炎上については、被害にあった当事者視点に基づいて検討した研究は稀少である。本稿では、オンライン・ハラスメントという概念を導入し、その現状を国内外の先行研究のレビューに基づいて概要を明らかにした。次に、Twitterで発生した女性記者ツイートの「炎上」事例を分析、ハラスメントを行うユーザーにはインフルエンサー群、インフルエンサーの犬笛に呼応する炎上加担ユーザー群、荒らしを行うユーザー群の三層があり、それぞれが異なる形で一人のユーザーに対してハラスメントを行っていたことを観察した。最後に、個人と組織が取るべき対策を述べる。", "keywords": [ "ソーシャルメディア", "オンライン・ハラスメント", "Twitter", "政策", "social media", "online harassment", "policy", "COVID-19" ], "content": "はじめに\n\n近年、デジタル技術が進展し、ソーシャルメディア1 (SNS) が普及することにより、多くの人が簡単に情報発信や収集を行うことが可能となった。また 2019 年に発生した新型コロナ感染症 (COVID-19) の感染拡大と、政府の緊急事態宣言を受け、できるだけ外出や対面を控えるため、人々がインターネットを利用する頻度も顕著に高まった（森下, 2021）。同時に、インターネット上のトラブル、とりわけSNS上におけるコミュニケーション不全に関する事件が多発している。いわゆる「ネット炎上」はもはや社会問題といっても過言ではない。「炎上」とは、ある人物が発言した内容や行った行為について、ソーシャルメディアに批判的なコメントが殺到する現象を意味する（山口, 2015）。\n\nしかしながら、炎上に関する既存研究は、SNS 上における情報の現象について扱うものが多く、とりわけ日本語環境の SNS については、炎上の被害にあった当事者視点に基づいて検討した研究は稀少である。そこで本稿では、炎上現象を検討する際に、オンライン・ハラスメントという概念を導入する。オンライン・ハラスメントとは、「インターネット上で行われる、扇情的なコメントやヘイトスピーチの繰り返しの投稿（「荒らし」「トロール」）、サイバーストーカー、身体的な脅迫、同意なしに性的に露骨な画像を公開すること（「リベンジポルノ」）、個人情報を公開する（「晒し」「ドクシング」）などの行為」を意味する (PEN America, n.d.-b)。\n\n本稿ではまず、オンライン・ハラスメントの現状を先行研究のレビューに基づいて概要を明らかにし、次に、日本で約 5,500 万人が使用する SNS である Twitter で発生した、女性記者ツイートの「炎上」、およびオンライン・ハラスメントの事例分析を行う (Statista Research Department, 2021)。最後に、個人と組織が取るべきオンライン・ハラスメント対策について述べる。\n\n\nオンライン・ハラスメントの現状\n\nオンライン空間において、どのようなハラスメントがどの程度行われているのであろうか。オンライン・ハラスメントは、デジタル技術や SNS が社会にもたらした負の影響であるといえるが、既存のデジタル技術に関する研究はこうした負の変化に楽観的であったことも関係して、オンライン・ハラスメントに特化した大規模な調査は多くはない (Fung et al., 2013; Jankowicz et al., 2021)。子どもの権利を推進する国際 NGO であるプラン・インターナショナルの 2020 年の調査によれば、日本の 15〜24 歳の若年女性 501 名のうち、25 %が「SNS で何らかの形でオンライン・ハラスメントを経験したことがある」と回答したという（プラン・インターナショナル, 2020）。他方、31 カ国で行われた調査結果を比較したところ、各国でオンライン・ハラスメントの概念について理解が異なること、回答者によっては全く理解されていないことも明らかとなった。\n\n米国・ピュー研究所による 2021 年の調査報告書によれば、米国人の 4 人に1人がオンライン・ハラスメントを受けたことがあり、このうち半数が政治的な意見を理由にハラスメントを受けたと答えた (Vogels, 2021)。また、2014 年、2017 年の同様の調査に比して、身体的な脅迫や、ストーキング、継続的なハラスメント、セクシュアル・ハラスメントなど、より深刻なオンライン・ハラスメントを受けたと答えた人数が増加したという。\n\n日本語環境については、インターネットプロバイダーの BIGLOBE 社による全国の 20 代〜60 代の SNS を利用する男女 770 人に対する調査において、SNS で他者から誹謗中傷2をされたことがあるかとの質問に対し、4.5%が「よくある」、13%が「たまにある」と返答した（BIGLOBE株式会社, 2020）。なかでも 20 代は年代別で最も顕著に誹謗中傷を受けた比率が高く、10%が「よくある」、18.9%が「たまにある」と回答したという。ただしここでの誹謗中傷はオンライン・ハラスメントの定義とは異なるため、注意が必要である。誹謗中傷とは、誹謗（他人の悪口を言うこと）と中傷（根拠のない悪口を言い、他人の名誉を傷つけること）の 2 つが合わさった語である（三省堂, 2013）。炎上とは、前述のとおり、誹謗中傷を含む批判的なコメントがソーシャルメディアに殺到する現象のことで、オンライン・ハラスメントは、誹謗中傷を含むハラスメント行為がオンライン上で行われることであり、時にその行為は集中的に行われ、炎上現象にもなり得る。\n\nオンライン・ハラスメントの全容を明らかにする試みとは別に、被害者の回復支援を優先したアプローチで行われた調査結果が存在する。文筆従事者の団体、国際ペンクラブの米国支部 PEN アメリカでは、記者やライターなどが SNS で深刻なハラスメントに遭っていることを重く見て、「オンライン・ハラスメントフィールドマニュアル」という調査研究事業を開始した (PEN America, n.d.-c)。同団体が 2017 年に実施した調査によれば、オンライン・ハラスメントを受けた 230 名から回答があり（うち 196 名は性別の明記があり、136 名が女性、52 名が男性、8 名がその他）、インターネット上で発生したオンライン・ハラスメントが、被害者の実生活に深刻な影響を及ぼしていることが明らかとなった。例えば、67%の回答者が「自分や身近な人の身の危険を感じたり、作品の発表を控えたり、SNS のアカウントを永久に削除するなど、深刻な反応を示した」、64.3%が「ハラスメントを理由に SNS を休止した」、62.3%が「オンライン・ハラスメントが私生活や身体的、心理的、精神的な健康に影響を及ぼした」と答えている (PEN America, n.d.-b)。また、ハラスメントを受けた理由としては、53.5%が「自分の政治的見解を述べたため」、「個人的な意見を述べたため」、38.9%が「性別や性自認を理由に標的にされた」、31%が「外見 (ルッキズム)」と回答しており、オンライン・ハラスメントが SNS 上で個人が意見表明を行ったこと、もしくは発言者のジェンダーやアイデンティティを理由に行われていることが分かる。\n\nまた、研究成果公開活動や、研究者同士のネットワーク拡大、また学生とのコミュニケーション促進の一助として、ソーシャルメディアの使用が奨励されている場合が多い大学界においても、研究者がオンライン・ハラスメントの被害に頻繁に遭い、有害な影響が出ていることが指摘されている。ゴッセら (2021)は、オンライン・ハラスメントは仕事、アイデンティティ、もしくは学者としての必要条件のように見なされているものの、ジェンダー、外見（ルッキズム）の要素が絡まることで複雑化していると述べた。\n\nソビエラージ (2020) によれば、オンライン・ハラスメントは誰でも被害に遭う可能性があるが、特に加害者の標的になりやすい属性があるという。すなわち、\n\n1) マイノリティである、\n\n2) フェミニスト（旧来のジェンダー価値観に縛られない）である、\n\n3) 政治、スポーツ、外交、防衛、サイバーセキュリティなど男性優位の領域について意見する\n\n場合、ハラスメントの標的になりやすく、これらの属性の共通部分においては最もハラスメントに遭う確率が高くなる（図 1）。なお、欧州の調査では、女性議員の 58.2%が SNS 上でオンライン・ハラスメントを受けたことがあると返答したが、特に年齢の低さ（40歳以下）、次に野党党員であることなど、議会政治において少数派である側面がハラスメントに遭うリスクを高めるとの結果であった（Inter-Parliamentary Union, 2018; 申・濱田, 2021）。\n\nオンライン・ハラスメントの被害に遭いやすい属性 (Jankowicz et al (2020)より).\n\n米国のオンライン・ハラスメント被害者の支援団体であるオンライン SOS による報告書によると、オンライン上のハラスメントはインターネットの商業化が進んだ 1990 年代から存在していたが、2014~2016 年の「ゲーマーゲート」3期を経て、2016 年米国大統領選挙において一連のハラスメントの流れが確立した(Lee, 2019)。すなわち、悪意をもつ行為者が、様々な方策を使用し、多数の媒体を通じて、複数の場所で、標的（被害者）に危害を与えるメカニズムが存在するのだという（図 2）。このように、最近の研究では、オンライン空間におけるハラスメント行為であっても被害者の受ける影響は甚大であること、またハラスメントや様々な暴力行為がオンライン空間ではなく、オフライン、すなわち現実世界に繋がることが問題視されている (Berry, 2019)。\n\nオンライン・ハラスメントの構成要素 (Lee, 2019) に基づく.\n\nこうしたオンライン・ハラスメントに対して、SNS プラットフォームはどのように対処しているのだろうか。ヤンコビッチら (2021) は、Facebook, Twitter, YouTube などユーザー数の多いSNSプラットフォームは、利用規約や「コミュニティ・ガイドライン」にハラスメント防止指針を取り入れているが、オンライン・ハラスメントの実態に比して画一的で、不十分であると指摘している。先述の PEN アメリカの調査においても、オンライン・ハラスメントを経験した 230 名のうち、53.1%がハラスメント被害をプラットフォームに通報したことがあると回答し、そのうち 70.8%がプラットフォーム側の対応は全く役に立たなかった、と回答している。\n\nこれらの既存研究は主に欧米の言語環境におけるオンライン・ハラスメントを分析したものである。そこで本稿では、事例分析を通じて、日本語環境におけるオンライン・ハラスメントの被害とメカニズムの一例を詳述することを試みる。\n\n\n事例分析 : X社の記者ツイート「炎上」事件\n\n本稿では、質的アプローチを用い、事例分析を行った。研究パラダイムとしては、解釈主義に基づく。分析対象とした Twitter アカウントは、大手メディア企業（以下X社とする）に所属する実名の女性記者のアカウント（以下Z氏）で、約 2 万のフォロワーを持ち、Twitter 社にも著名人であるという認証を受けている。筆者は、Twitter を使用し、従前より同アカウントをフォローしており、そのため同アカウントが以前に「炎上」の被害を受けていたことを確認していた。分析対象とした事例は、新型コロナウイルスによる死者数の多寡に関するツイートの掲載を発端とする炎上であるが、本ツイートがなされた時期は日本でも過去最多の新規感染者数が記録されていた頃であった（日本経済新聞, 2021）。このことも関係してか、後述するとおり、本ツイートは多数のユーザーによって引用を含むリツイートがなされ、筆者らの目にも入ることとなったため、事例分析の対象として選択した。\n\nZ 氏は、2021 年 8 月 3 日午後 7 時 12 分に、新型コロナ感染症拡大による死者の数について、これを少ないと表現するツイート等を見ると悲しくなる、数の多寡にかかわらず、亡くなった人一人ひとりに人生があり、それを「少ない」などと特に著名人が矮小化するのはいかがなものか、という内容のツイートをした。\n\n本ツイートは掲載後、約 3 日間にわたり批判的なツイートが集中し、炎上状態となった。本稿では、2021年 8 月 3 日〜7 日の間にZ氏アカウントに直接届いた 2,817 件の直接リプライ（直接リプライ1,175 件、これらのリツイート1,499 件、引用リツイート143 件）、及び 5,595 件の本ツイートの引用リツイート（以下引用 RT）の分析を行った。データ分析対象ツイートは、ExportData.io サービスを利用し、（to:[Z 氏のアカウントID]） until: 2021-08-07 since: 2021-08-03 および Query: https://twitter.com/[Z氏のアカウントID]/status/1422500544032038919, Result type: mixed, Language: ja で 2021 年 8 月 7 日に取得した。なお、同等の機能を果たす無料の代替品には、Twitter の公式 API (https://developer.twitter.com/en/products/twitter-api、ないしhttps://developer.twitter.com/en/docs/twitter-api/tools-and-libraries) がある。\n\nなお、直接リプライを中心に分析を行うのは、仮に引用RTが拡散されても、被引用ユーザーには通知が届かず、静観できる状況であるものの、被引用ユーザーに対して直接リプライ、もしくは引用 RT がなされると、本人に通知が届き、とりわけそのコメントが被引用ユーザー本人のアイデンティティ等、元ツイートの内容以外について批判的なコメントであった場合、ハラスメント行為に直面することとなり、精神的苦痛が大きいためである。\n\n図 3 はZ氏アカウントへの直接リプライ及び直接リプライをリツイート、引用 RT したツイートの時間当たりの数をグラフ化したものである。これからは、一時間あたり直接リプライの数が、8 月 3 日午後 11 時台に一度ピークを迎え、低下した後、翌 8 月 4 日午後4時台から再び急増し、午後9時台になるまで、一時間あたり優に 100 近くのリプライが届く状態が継続したことが分かる。質的分析ソフト NVivo Windows （2020 年 3 月リリース版）を使用して、直接リプライの感情を自動で分類したところ、図 4 のようになり、大部分の直接リプライが批判的な内容であることが判明した。なお同等の機能を果たすフリーの代替品として、KH Coder 3 や、Python のライブラリML-Ask がある。これらを用いることで、ツイートに現れる単語の出現頻度や感情を分析することができる。\n\n一時間あたり直接リプライおよびそれらのリツイート、引用 RT 数.\n\n感情の自動コーディングの結果.\n\nさらにツイートを手動で分類したところ、1,175 件の直接リプライのうち、肯定的な内容は 147 件、批判的な内容は 1,022 件となっていた。また、同ソフトを使用してワードクラウドを作成したものが、図 5 である。図からは、直接リプライを送った者は「日本における（新型）コロナ（ウイルス感染症）による死者数は少ない」という旨を主張していることが分かる。\n\nワードクラウド (固有名詞は匿名化).\n\n直接リプライを手動で分類したところ4、表 1 のとおりであった。最も数の多かった元ツイートの内容に対する主張は、「（死者数が）少ないという表現は妥当である」というもので（216 件）、次は「（新型コロナ感染症の）他にも重要な事柄がある」という内容のものであった（170 件）。これらは、形式的誤謬ないし偽善の抗弁である「そっちこそどうなんだ主義 (whataboutism) 」に基づくコメントである。また、71 件の直接リプライは、元ツイートが扇動的であると見なし、批判を行っていた。\n\n直接リプライの内容の分類と件数.\n\n直接リプライは、元ツイートの内容ではなく、Z氏の属性に対して批判もしくは誹謗するものも多かった。例えば、「恥を知れ」「心の底から軽蔑する」といった、元ツイートの内容には全く関係のない個人攻撃が 195 件、X社批判が 186 件、マスコミ批判が 177 件、「感情的になるな」といった女性差別的なコメントが150件あった。このように、Z氏がオンライン・ハラスメントを受けた理由としては、Z氏が政治的見解を述べたこと、個人的な意見を述べたこと、女性であること、外見（ルッキズム）、という PEN アメリカの調査結果でも見られた、SNS 上で個人が意見表明を行ったこと、もしくは発言者のジェンダーやアイデンティティを理由に行われていることが分かる。これに加え、日本語インターネット空間独自のアイデンティティ攻撃の理由として、Z氏がX社の記者であるという側面があることが分かった。\n\n次に、Z氏に対する直接リプライと、元ツイートの引用 RT の関係を分析したところ、炎上を通じてハラスメントを行ったユーザーは、3 つの層に分かれることが見て取れた。すなわち、①インフルエンサー（高頻度炎上関与ユーザー群）、②インフルエンサーの「犬笛」に呼応する炎上加担ユーザー群、③荒らしを行うユーザー群、である。\n\n小山ら(2019) による計約 13 万人の炎上参加ユーザーの分析によれば、炎上参加者は他の炎上にも参加しやすいことが明らかになっている。5 件以上の炎上に関わったユーザー77 名に関する詳細な分析においては、それらのユーザー間にはフォロー/フォロワー関係が密に存在し、高頻度炎上関与ユーザーは多くのフォロワー数を持ち、同じ炎上トピックに対してツイートを行うという、情報の共振構造が確認できたという。Z氏の炎上事件においても、多くのフォロワー数を持ち、特定の政治傾向を持つユーザーが、フォロー/フォロワー関係を結び、ユーザー間でZ氏のツイートの引用RTを順次行っていた（図 6, 図 7）。これらユーザー群が他の炎上に関わっていたかは本稿では確認できず、高頻度炎上関与ユーザー群とみなすことは難しいが、多くのフォロワー数を持つユーザーの引用 RT が、フォロワーを通じて広くTwitter 全般に拡散されていったことが推測できる（図 8）。\n\n一時間あたり直接リプライとインフルエンサーの引用 RT.\n\nインフルエンサーのフォロー/フォロワー関係 (カッコ内フォロー数、フォロワー数).\n\nインフルエンサーによる引用RT拡散ネットワーク図.\n\n②のインフルエンサーの「犬笛」に呼応するユーザー群であるが、犬笛とは「加害的もしくは有害な意味を持つ二重、もしくはコード化された言葉や記号を使い、インターネット上の加害者グループに特定の標的を攻撃するように合図する行為」を意味する (PEN America, n.d.-a)。先のインフルエンサーのうち、約350 万ユーザーをフォロワーに持つH氏は、Z氏のツイートを2度に渡って引用 RT を行っている。第一回目の引用 RT は、2021 年 8 月 4 日午後 6 時 9 分に行われ、記者は新型コロナウイルスによる死者数は多いと読者の恐怖を煽ることで販売部数を稼ぐ最低のビジネスをしている、もっと科学を勉強して冷静かつ論理的に記事を書いてほしいが、無理であろうから当該メディア自体なくなってほしい、という内容であった。第二回目の引用 RT は、同日午後 7 時 11 分に行われ、Z 氏によるツイートは新型コロナウイルスに対する恐怖を煽って儲けることを正当化しており、最低である、という内容であった。\n\nこの 2 つのツイートに共通する主張が「煽るな」というものである。先述のとおり、直接リプライのうち、元ツイートが扇動的であると批判したものは71件であったが、これらの発生した時間帯を時系列で確認すると、それまでは合計 6 件しかこの内容の直接リプライがなかったものの、H 氏が一度目の引用 RT をした午後6時台は7件、二度目の引用 RT を行った午後7時台は 15 件、午後8時台は11件と、急増している（図 9）。また、H氏のツイートを見て来たと述べたツイートも3件あり、インフルエンサーの犬笛に呼応して、わざわざZ氏のアカウントに対して直接、故意の攻撃を行っていることが見て取れた。なお、山口 (2015, 2016, 2020) による炎上現象の既存研究によれば、炎上加担の理由として正義感をあげる人が大半とあるが、本ユーザー群はこうした大多数の炎上加担者であることが推測される。\n\n「煽るな」という内容の一時間あたり直接リプライおよびインフルエンサーの引用リツイート時間.\n\n直接リプライの分析からは、インフルエンサー、及びインフルエンサーの呼びかけに直接・間接的に反応したと推測されるユーザーとは別に、③荒らし (扇情的なコメントやヘイトスピーチを繰り返し投稿すること)を行うユーザー群が見て取れた。まず、195 件の個人攻撃のツイートのうち、12 名のユーザーが 2 度以上、繰り返し個人攻撃のリプライをZ氏に送っていた。また、図 10、図 11 は批判的な内容の直接リプライ、及び個人攻撃のリプライの一時間あたりの数をグラフ化したものであるが、両者の数量は必ずしも呼応していない。特に一時間あたりの個人攻撃コメントが多かった時間帯（2021 年 8 月 4 日午前 11 時〜8 月 5 日午前 2 時）について、先のインフルエンサーの犬笛に呼応した批判的ツイートの一時間あたり数と比較すると、特に増減傾向に類似性が見られない（図 12）。これらから推測されるのは、常日頃から Z 氏の発言をモニターし、期を見て荒らしを行うユーザー群が存在する可能性である。実際、自称メディアウォッチャーで7千余のフォロワーを持つユーザーが、2021 年 3 月 18 日に、X社には一部、低質かつイデオロギー色の強い記者がいる、という内容のツイートを行い、X 社の「低品質」記者一覧として、Z 氏ほか複数名の X 社に関係する記者の Twitter アカウント情報を掲載している。なお、こうしたユーザーが、Z 氏の他のツイートに対しても繰り返し荒らしを行っているか、またいかなる動機に基づいて荒らしを行っている・行ったのかはさらなる調査分析が必要である。\n\n一時間あたり批判的コメントと個人攻撃コメント.\n\n一時間あたり批判的コメントと個人攻撃コメント.\n\n「煽るな」コメントと個人攻撃コメントの比較.\n\n\nオンライン・ハラスメント対策\n\n国内外の既存研究から、誰であってもオンライン・ハラスメントを受ける可能性があること、一方でハラスメントを受けやすい属性があること、ハラスメントの被害は身体的・精神的に甚大であることが明らかとなった。また、日本語環境の Twitter における事例として、X 社の記者ツイート「炎上」事件を分析し、オンライン・ハラスメントを行うユーザーにはインフルエンサー群、インフルエンサーの犬笛に呼応する炎上加担ユーザー群、荒らしを行うユーザー群の三層があり、それぞれがなる形で一人のユーザーに対してハラスメントを行っていたことが見て取れた。このようなオンライン・ハラスメントに対して、ユーザーはどのような対策を取ることができるのだろうか。\n\n各国の女性議員へのオンライン・ハラスメントの調査分析を行ったヤンコビッチら (2021) は、報告書に政策提言を記載している。具体的には、SNS プラットフォーム企業への提言として、\n\n• 個別ツイートの通報ではなく、インシデント報告制度の導入\n\n• プラットフォームから各企業・団体の SNS 担当者への情報共有\n\n• 利用規約の適用の徹底\n\n• ナッジ機能の強化\n\n• プラットフォーム間のオンライン・ハラスメント防止機構の設置\n\n政策担当者への提言として、\n\n• SNS 規制の強化（透明性のある報告システム、プラットフォームの報告義務）\n\n• ディープフェイクなどに加え、ジェンダー化されたハラスメントも規制対象に含めるべき\n\n• 米国で1994年に制定された「女性に対する暴力阻止法案」に、物理的な危害だけでなくオンライン上の危害も含めるべき (Congress.gov, 1994)\n\n雇用者への提言として、\n\n• 被雇用者の SNS 発信に関するガイドラインの設定\n\n• 心理的・道徳的・金銭的な支援。オンライン・ハラスメント被害者の晒し防止対策のためのサービス料の支払い、プラットフォームへの被害報告代行\n\nを挙げている。しかし、日本語のインターネット環境の場合、オンライン・ハラスメントに関する社会的認知度が低い上に、主に外国に本社を置くプラットフォーム企業の対策も十分とはいえないため、ハラスメント防止策はユーザー個人、もしくは個人が所属する組織が取ることが求められているのが現状である。以下、日本語SNS環境の特殊性を念頭に置きつつ、個人および組織がとり得るオンライン・ハラスメント対策について、Twitter を中心に述べる。\n\nハラスメント対策の最も基本的な行動としては、ミュート（特定のアカウントのツイートがタイムラインに表示されないようにする Twitter の機能）、ブロック（特定のアカウントによるメッセージの送付、ツイートの閲覧、フォローの禁止）、通報（攻撃的なツイートまたはアカウントを Twitter 運営に報告すること）の 3 つがある (PEN America, n.d.-d). また、いつどこでどのようなハラスメント被害を受けたのか、ログを取っておくことも重要である。こうした情報は、オンライン・ハラスメント行為を警察や弁護士に相談する際、重要な証拠となる（表 2）。\n\nハラスメント対応ログ.\n\nまた最近では、オンライン・ハラスメント対策の専用ツールが開発されており、こうしたツールの導入も間接的なハラスメント防止になり得る。例えば Block Party というツールは、誰からの返信が見たいか、見たくないかをフィルターとして設定することで、基準に該当する返信を全て自動でミュートする。これにより、ユーザー自身は Twitter を開いてすぐに嫌がらせのツイートを目にするというようなことを経験せずとも、自分自身で後日、もしくは信頼できる人物に依頼して返信の内容を確認することが可能になる (Cho, n.d.)。さらに、Twitter 社が、「安全モード」機能を検討中である旨が発表された (Doherty, 2021)。本機能を有効にすると、自分に対して有害な言葉を使ったユーザーや、荒らしを行ったユーザーが自動的に7日間ブロックされるという。その他、全般的なデジタル衛生のチェックや、SNSアカウントのチーム運用も対策として考えられる (Jankowicz et al., 2021)。\n\n昨今は、議員、記者、研究者など既存メディアを通じて従来発信してきた者でなくても、SNS を通じて発信し、広く社会にアウトリーチすることが求められている。またZ氏の例では、Z 氏がX社に所属することを理由にハラスメントが行われていた。炎上やオンライン・ハラスメントは個人アカウントのみならず、大小様々な組織に対しても発生している（榊・鳥海, 2020）。したがって、所属員の SNS アカウント運用とそれに伴うハラスメント被害について、組織が全く関与しないことは難しい。\n\n組織ができる対策として、まずは炎上決着メカニズムを理解することが不可欠である。田代・折田 (2012) は、ネット炎上を「不具合」に対して決着をつけようとすること、と見なし、炎上が収束するためには「決着」が必要とした。そして、ハラスメントが収束するためには攻撃側が納得することが必要であり、法的に問題があれば法的に処理されることで収束に向かう（図 13）。謝罪を行ったとしても、単に謝罪しただけでは収束せず、謝罪が受け入れられて収束する。炎上が議論へ繋がって収束する、コメント欄やブログそのものの削除 (Twitter の場合はリプライ機能の制限や Twitter アカウントの削除)、炎上を無視し、忘れ去られることで収束を迎えることもある、とする。所属員の SNS アカウントが万が一炎上したり、ハラスメントの被害者となった場合、そうした行為を受けたことを批判したり懲罰の対象とするのではなく、収束には様々なパターンがあることを理解することが、SNS 運用者支援の第一歩であろう。\n\n決着のフロー (Twitter を想定、田代・折田 2012) に基づく.\n\n第二に、組織がインターネット上でどのような属性を与えられているか、それに基づく荒らしユーザーの確認が求められる。事例分析ではメディア企業である X 社の例を扱ったが、他にも例えば大手消費財化学メーカーである花王は、韓流ドラマをよく流すフジテレビへのスポンサーを多くつとめていたことから、「韓国に貢献し日本文化を破壊する企業である」という誤情報が拡散し、インターネット上で批判を受けた（田代・折田, 2012）。先述のとおり、オンライン・ハラスメントには加害者の標的になりやすい属性の集合があり、集合の共通部分においては最もハラスメントに遭う確率が高くなる。企業が自社のブランド認知度を調査するのと同様、ハラスメントの標的となる可能性についても検討する必要があるだろう。\n\n先に、個人ではミュート、ブロック、通報が最も基本的かつ効果的なハラスメント対策であると述べた。他方で、議員、記者、公的組織の所属員によるアカウントなど、発信する情報に公共性があると見なされる場合、ブロック行為が好ましくないと見なされるケースもある（日本経済新聞, 2021）。したがって、組織内でブロック機能の利活用について議論の上、ガイドラインを設け、適切に運用することが求められる。仮に所属員が自己防衛としてとり得る有効策を、所属組織が禁じるようであれば、所属員がハラスメントを受け身体的・精神的ダメージを受ける可能性も高まるのであるから、より充実した心理的・道徳的・金銭的な支援が必要となる。\n\n日本では、2021 年 4 月 21 日、インターネット上で誹謗中傷の投稿を行った人を特定しやすくするためのプロバイダー責任制限法の改正案が可決され、加害者の特定にかかる手続きが簡素化された。また、10 月 7 日には、刑法の「侮辱罪」に懲役刑を追加する法改正の要綱案が、法制審議会の部会により取りまとめられ、法務大臣に答申するとの報道がなされている（共同通信, 2021）。それでも開示手続きなど、オンライン・ハラスメントの直接的・間接的費用の負担は加害者よりも被害者にとって重いことは変わらない。したがって、例えば、弁護士保険の費用一部負担といった形で、組織が所属員への金銭的支援を行うことを検討すべきであろう。\n\n\nおわりに\n\n国内外の先行研究から、誰であってもオンライン・ハラスメントを受ける可能性がある一方で、ハラスメントを受けやすい属性があること、また、ハラスメントはオンラインで行われるものであっても、被害者が受ける身体的・精神的ダメージは看過できないことが明らかとなった。日本語環境の Twitter におけるオンライン・ハラスメントの事例として、大手メディア企業X社の記者ツイート「炎上」事件を分析した結果、ハラスメントを行うユーザーにはインフルエンサー群、インフルエンサーの犬笛に呼応する炎上加担ユーザー群、荒らしを行うユーザー群の三層があること、さらに、それぞれが異なる形で一人のユーザーに対してハラスメントを行っていたことが観察できた。事例分析は炎上事例の一例でしかないため、一般化は難しいが、SNS 上の炎上現象に関する先行研究の結論を補完するものである。本稿で導出した仮定に基づき、より大規模な分析を行うことを将来の課題としたい。\n\n本稿の最後には、日本語のインターネット環境の場合、オンライン・ハラスメントに関する社会的認知度が低いことや、Twitter など外国に本社を置くプラットフォーム企業に対策を求めることの難しさを鑑み、SNS ユーザー個人、もしくは個人が所属する組織が取るべきハラスメント防止策を詳述した。日本語環境の SNS については、オンライン・ハラスメントという概念自体が知られていない状況であり、その社会的影響に比して、ハラスメントにあった被害者を中心に検討した論考が少ない。こうした意味で、本稿が学術的な議論の広がりと深化の一助となり、実効的なハラスメント防止策に繋がることを期待したい。\n\n本論文の研究結果の基礎となるデータは，すべて本論文中に示されており，追加のソースデータは必要とされていない。例外として、ソーシャルメディアのデータには倫理上および著作権上の制限があるため、本研究の基礎データを共有することはできない。対象となるデータは、2021 年 8 月 3 日に投稿された大手メディア企業に所属する女性記者（以下Z氏）のツイートと 2021 年 8 月 3 日 ~7 日の間に Z 氏アカウントに直接届いた 2,817 件の直接リプライ（直接リプライ1,175 件、これらのリツイート1,499 件、引用リツイート143 件）、及び 5,595 件の本リツイートの引用リツイートである。本研究の再現を可能にする詳細な情報は、「事例分析」部分に記載されている。方法に関する質問がある場合は、著者 (tonami.aki.ka@u.tsukuba.ac.jp) に連絡されたい。\n\nTweet ID についても、Z 氏の匿名性を守るためのセキュリティ上の配慮から共有しない。同分野の研究者が、善意と明確な研究目的をもってIDの閲覧を希望する場合、データの利用目的と方法を明記の上、著者 (tonami.aki.ka@u.tsukuba.ac.jp) に連絡されたい。\n\n本稿の執筆にあたり、M氏には荒らしユーザーや個人でできるオンライン・ハラスメント対策について助言をいただいた。心から感謝申し上げます。", "appendix": "参考文献\n\nBerry E: Online violence just as destructive as offline violence.2019. 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(accessed 13 September 2021d).Reference Source\n\nSobieraj S: Credible Threat: Attacks Against Women Online and the Future of Democracy. Oxford: Oxford University Press; 2020.\n\nStatista Research Department: Leading countries based on number of Twitter users as of July 2021. (accessed 7 October 2021).Reference Source\n\nVogels EA: The State of Online Harassment. Pew Research Center; 13 January 2021. Reference Source\n\n共同通信: 「侮辱罪に懲役刑」厳罰化答申へ.2021. (accessed 7 October 2021).Reference Source\n\n榊剛史, 鳥海不二夫: ソーシャルポルノ仮説に基づくメディア企業SNSアカウントの扇動性に関する分析. The 34th Annual Conference of the Japanese Society for Artificial Intelligence. 2020.\n\n三省堂: スーパー大辞林.2013.\n\n申琪榮, 濱田真里: 女性議員に対するオンラインハラスメント—首都圏の女性地方議員の事例を手がかりに—. 生活経済政策. 2021; 291: 19–23.\n\n田代光輝, 折田明子: ネット炎上の発生過程と収束過程に関する一考察〜不具合に対する嫌がらせと決着による収束〜. 情報処理学会研究報告. 2012.\n\n日本経済新聞: 東京都、新たに5386人感染過去2番目の多さ 7日平均で前週の117.9%.2021. (accessed 20 October 2021).Reference Source\n\nプラン・インターナショナル: 若年女性へのジェンダーを理由にしたオンライン・ハラスメントに関する調査結果日本の調査報告書.2020 October. Reference Source\n\n森下真理子: 自宅でのモバイルネット利用が増加!コロナ禍が浮き彫りにした情報メディアニーズ.2021. (accessed 22 September 2021).Reference Source\n\n山口真一: 実証分析による炎上の実態と炎上加担者属性の検証. 情報通信学会誌. 2015; 33(2): 53–65. Publisher Full Text\n\n山口真一: 炎上加担動機の実証分析. 2016 年社会情報学会(SSI) 学会大会. 2016. Reference Source\n\n山口真一: 正義を振りかざす「極端な人」の正体. 光文社; 2020.\n\n\nFootnotes\n\n1 個人、コミュニティ、組織が、簡単にアクセスできるユーザー生成コンテンツを作成、共同作成、修正、共有、関与することを可能にすることで、共同作業、接続、相互作用、コミュニティの構築を可能にする Web ベースのサービスのこと (McCay-Peet & Quan-Haase, 2017)。\n\n2 同調査では、「根拠のない悪口を言いふらして他者の名誉を傷つけたり、人格を否定するような言葉で他者を傷つけること」と定義されている。\n\n3 2014 年夏頃から欧米で発生した、表現の自由を掲げるゲーム愛好者と、女性差別の観点から表現規制を求めるフェミニストらの対立がインターネット上で激化した事件。特定の女性プログラマーに対し、個人情報の流出、殺害予告、脅迫を含む過激なオンライン・ハラスメントが行われた。\n\n4 同一ツイートに複数のコードが含まれる場合もある。" }	[ { "id": "100519", "date": "09 Dec 2021", "name": "Yasuko Kawahata", "expertise": [ "Reviewer Expertise SNS", "社会情報", "メディア社会" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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イントロダクション・前文では、オンライン・ハラスメントに関しての発生起因における最新の動向に関する研究、事例紹介などの論考が掲載されており、一般の方々を対象とした昨今のオンライン上におけるリスクマネジメントに関する議論がまとめられているコレクティブ・インフォメーションとしての役割を果たすだろうと考えました。\n（1）全体のレビュー一方で、今回の記事はジャーナリストを対象にした比較的顕著な一例における事例であるかとも存じます。そういった面では、多くの課題が示唆できる論文でもあり、今後も議論が続くであろうオンライン・ハラスメントにおける問題解決の活路を提示する位置づけの論文としての役割も果たしていると考えております。今回の議論ではジャーナリスト（Z 氏）の一例を元に議論を展開していきましたが、広範なメディア単位でも表出しつつある課題でもあり、ミクロな観点でも一般のコミュニティ内でもソーシャル・ハラスメントとしてオンラインが利活用されるリスクは存在するため今後も議論を要する課題であると考えます。本稿では、分析対象としたSNS上のソースデータに関して被害者・加害者の両者のプライバシーに配慮をした考察がされている点に関しても（編集部了承済み）、今後の本議論の展開の方法論に関しての示唆も提示されており、より議論の深化に寄与されればと考えます。\n（2）追論すべきポイント本稿に関連するトピックとして、2021年11月31日にプレスリリースされましたが、Twitter上におけるなりすましに関するポリシー・利用規約が改訂されたことが今後、本議論でもどのように取り扱われるか、有効に働きうるかは課題です。 “Twitter Safetyは「女性、活動家、反体制派、マイノリティコミュニティの一員に対し、過度に影響を与える可能性がある」とし、ポリシーに違反する画像や動画がシェアされた場合、Twitterはそのメディアを削除するとともにしかるべき強制的対応、たとえば当該ツイートを検索結果に表示されにくくしたり、ツイートの削除対応をしたうえで投稿者に通知し、またTwitterはポリシーに違反しているユーザーアカウントを永久に凍結する権限もあるとしています。” 意訳・引用元：https://twitter.com/TwitterSafety, 参照：2021/12/02) といった具体的な対策に関するケースも言及され、より一層本稿における議論は加速化すると考えております。主に、本稿でも示唆されている通り、ある“特定の個人”に不特定多数で、攻撃的な行為を行う、「成り済まし」を行った上で誹謗中傷を「暗に行う」事例などといった、陰湿なソーシャル・ハラスメント行為、所謂「ソーシャルポルノ」の事例も観測されております。また、搾取対象に対して無許可にメディア化する行為、身体的特徴を揶揄する行為、反社会的行為、つまりは個人の利益、日常行為を著しく害する行為などもSNS上で見受けられており、上述の引用のように注意喚起なども情報共有されつつある点ではプラットフォーム単位での環境改善は始まっているものの、まだこれから課題解決に働きかけが必要となる要素が多くあります。\n（3）本議論に関する今後の課題点、調査展望に関して本稿における課題に関しては、オンライン・ハラスメントの手法がテキストなど文面・ショートメッセージで発露するものから、トラッキングが困難になりやすい画像、イラスト、映像でのソーシャルポルノの暴露なども目立ってきている観点もあります。そのため、本論のようなケースに関して、さらに注視、また注意喚起などが教育機関での情報共有もなされるべきだと考えております。特に画像・音声メディア・映像など拡散性の高いメディアとなるとさらなるソーシャルポルノのリスクが発生し、被害者・加害者周辺における事実確認などが困難にもなりやすく隠蔽工作なども進みやすいところで、泣き寝入りとなるケースに関してもSNS上で被害者の声として観測されております。そのため、まずは問題意識の共有の契機は必要であると考えます。（上記のケースに関してはレビュワーによる匿名調査の元での見解。）そういった面で、学術的価値がある内容であると考えます。今後、より対策に講じるべき課題であり、これら問題解決をする際のSNS上での議論の過熱化、また加害者・被害者に対する合理的配慮、紛争解決の議論などを始めていくべきでもあり、インターネット上の違法・有害情報に対する対応（プロバイダ責任制限法）の改正なども本年度行われた観点も踏まえ、より学術的な議論も展開できればと考えるところです。また、本議論を展開するにおいてSNS上における「リスクマネジメント」など教育関係者での情報共有など、唐突な有害情報の暴露のリスクまで考慮にいれた議論、対応などもより必要なるかと考えます。そういった面で、本論における議論・論文は情報共有をする面でも有益に働く内容かとも存じます。\n（4）まとめ本稿でも触れている通り、ハラスメント防止策に関する提案に関して提示しているところ、インシデントの事例収集も必要になっていると考えます。特に、裁判など起こす力、知識を持たない若年層、または弱者など搾取対象とされやすい対象に対するインシデントは今後も発生しうると考えるため、これらの問題収集、対策の議論など情報共有、コミュニティ形成を推し量っていく必要もあるかと存じます。特に海外サーバーにあるプラットフォームにおけるインシデント・紛争解決は論文中でも触れてあるように解決までに時間を有することが多く、実効的な防止策に関する議論に関して継続的な試み、論文などにおけるジャーナリズムとしての意見共有の機会も必要かと考えるところ、本稿が一つの議論の深化に接続すればと願っております。\n参考文献 Twitter, Inc.: なりすましに関するポリシー. n.d. available at: https://help.twitter.com/ja/rules-and-policies/twitter-impersonation-policy. Accessed 2021/12/02. 総務省: インターネット上の違法・有害情報に対する対応（プロバイダ責任制限法）. n.d. available at: https://www.soumu.go.jp/main_sosiki/joho_tsusin/d_syohi/ihoyugai.html\n\n本研究は明確かつ正確に提示されたものであり、最新の文献を引用していますか。はい\n\n研究設計は適切で学術的価値がありますか。はい\n\n方法と分析について第三者による再現が可能となるよう十分な詳細が提示されていますか。はい\n\n（該当する場合は要回答）統計分析および解釈は適切ですか。対象外（統計を使っていない\n\n結果の基礎となるソースデータはすべて入手可能で再現性を十全に保証していますか。ソースデータは不要\n\n結論は結果により妥当な裏付けを得ていますか。はい", "responses": [ { "c_id": "7699", "date": "03 Feb 2022", "name": "Aki Tonami", "role": "Author Response", "response": "川畑先生査読コメントをいただき、誠にありがとうございます。いただいたコメントは、どれも大変重要、かつ今後の研究の発展に向けて参考になる指摘であると存じます。(2)(3)の追論のポイントに関しましては、今後の課題として、クロスプラットフォームで行われるハラスメントや、なりすまし、テキスト以外を使用したハラスメント行為の検討が必要として、追記いたしました。" } ] }, { "id": "100521", "date": "20 Dec 2021", "name": "Shinichi Yamaguchi", "expertise": [ "Reviewer Expertise 計量経済学", "ソーシャルメディア", "社会情報学", "情報経済論" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n本研究では、オンライン・ハラスメントという概念を導入し、その概要を明らかにすると共に、Twitterで発生した炎上事例を分析してハラスメントを行うユーザを整理しています。また、最後には個人と組織がとるべき対策について詳細に述べられています。著者らが指摘するように、炎上の被害に遭った当事者視点に基づいて分析した研究は少なく、そこにオンライン・ハラスメントという概念を導入して整理・分析を行ったことは意義が高いといえます。実証分析も丁寧で、社会の関心も高い分野といえ、研究成果に関心のある読者も多いでしょう。　しかしながら、これを学術論文として考えた時に、いくつか問題点を抱えていると思います。具体的には、以下の点に改善出来るところがあると思います。\n①既存研究レビュー・分析・対策の関連性がない　最も大きいのが、各節の関連性がなく、報告書のように知見を羅列しているところだと思います（各知見自体は非常に有意義なものですが、学術論文としては課題があるという意味です）。例えば、大きく紙幅を割いている図2について、「悪意をもつ行為者が、様々な方策を使用し、多数の媒体を通じて、複数の場所で、標的（被害者）に危害を与えるメカニズムが存在する」と指摘している一方で、分析されているのはTwitterのみです。さらに、結構な分量を割いている「オンライン・ハラスメント対策」も実証分析と全く関係のない記述が延々と続いています。せっかくハラスメントユーザのパターンを明らかにしたのに、活かされていません。　その要因の1つに、節の構成があると思います。まず、序盤では「はじめに」と「オンライン・ハラスメントの現状」とあります。後者は既存研究レビューといえますが、本当に現状を述べるのが中心となっており、既存研究にはどのような課題があり、そのために本研究では～を明らかにするという明確な研究の問いが示されていません。強いて言うなら「欧米の言語環境の研究しかないので日本語環境での一例を記述する」というところがそれに該当すると思いますが、もう少し本研究の新規性・社会的意義を示すためにも、既存研究との位置づけとResearch Questionをはっきりさせた方が良いと思います。そのためには、既存研究よりも、本実証分析の内容に関連するものが必要だと思います。具体的にいうと、オンライン・ハラスメントの攻撃者を分析した研究です。Research gapやResearch Questionについて、別途節を設けても良いかもしれません。　また、事例分析の節では手法と分析結果が一緒の節に入っているので、分けて書くと同時に、手法をもっと詳しく書いたほうが良いと思います。特に、本研究で重要な知見である3つの層の分類について、「元ツイートの引用RTの関係を分析」としか書かれておらず、手法が分かりませんでした。　そして、得られた結果と先行研究との関係を議論する節や、結果から導かれる対策を論じる結論節を用意したほうが良いと思います。現在の「オンライン・ハラスメント対策」で書かれている内容は興味深いのですが、これらは縮小させ、分析から得られる対策の検討に紙幅を割くことを推奨します。\n②事例が1つしかない　1事例をもとにオンライン・ハラスメントのパターン分析をするのは、やや無理があるように思いました。事例分析なので数は多くなくて良いと思いますが、頑健性を検証するためにももう2，3事例の分析があった方が良いと思います。ただし、これは難しいと思うので、修正要求ではなく修正提案となります。\n③その他（細かい点）・分析において、「「感情的になるな」といった女性差別的なコメント」とありますが、「感情的になるな」単体で女性差別的と解釈してよいのか疑問が残りました。女性差別的に使われることが多いのは理解していますが、男性相手にも使う言葉だからです。また、「心の底から軽蔑する」が個人攻撃に入るのならば、「感情的になるな」も個人攻撃なようにも思います。・小山（2019）が文献リストに見つかりませんでした。\n\n本研究は明確かつ正確に提示されたものであり、最新の文献を引用していますか。はい\n\n研究設計は適切で学術的価値がありますか。一部該当\n\n方法と分析について第三者による再現が可能となるよう十分な詳細が提示されていますか。一部該当\n\n（該当する場合は要回答）統計分析および解釈は適切ですか。一部該当\n\n結果の基礎となるソースデータはすべて入手可能で再現性を十全に保証していますか。ソースデータは不要\n\n結論は結果により妥当な裏付けを得ていますか。一部該当", "responses": [ { "c_id": "7700", "date": "03 Feb 2022", "name": "Aki Tonami", "role": "Author Response", "response": "山口先生査読コメントをいただき、誠にありがとうございます。 ①既存研究レビュー・分析・対策の関連性がない本ジャーナルの研究論文の前提として「Research Articles should present originality in findings and insights and offer theoretical, empirical, experimental and/or methodological advances to their respective fields of research. Null and negative findings and reanalyses of previous studies leading to new results, as well as confirmatory results, are also encouraged.」とあります（https://f1000research.com/for-authors/article-guidelines）。研究の独自性を提示するに際し、どの程度先行研究のレビューに紙幅を割くべきかは分野によって異なりますが、本稿ではあえて、オンラインハラスメントが言語、文化、文脈に高度に依存することから、主に英語による、英語圏におけるオンラインハラスメントに関する先行研究の網羅的レビューを避け、そもそも存在していない日本語環境におけるオンラインハラスメントの論考の第一号となることを目指しました。したがって、Research gapは既存研究がないこと、となります。また本稿は質的研究であり、実証や仮説検証を目的とするものではありません。よって、Research questionは「オンライン・ハラスメントの現状はどうなっているか」という記述的問いになります。ただしこれが分かりにくいとのご指摘かと存じますので、その旨加筆修正いたしました。なお、本論文が発表された後に発表されたオンラインハラスメントに関する日本語の研究では、例えば、法学セミナー2021年12月号特集『言論に対するゆるしと制裁』が参考になるかと考えます。「学術論文としては課題がある」とのご指摘については、想定されている学術論文が具体的にどのような論文なのか不明なため、前提と求められている条件を明示していただければ幸いです。（例えばIMRaDの形式で書かれることの多い量的研究の論文なのか、など。）図2において、オンラインハラスメントがTwitter以外の複数のプラットフォームに渡って行われていると述べているのはご指摘のとおりです。しかしながら、マルチプラットフォームにおけるハラスメントについては本稿の対象範囲外ですので、今後の課題として脚注を追加いたしました。手法を「もっと詳しく書いたほうが良い」とのこと、「元ツイートの引用RTの関係を分析」について詳細を記述しました。もし、その他にも不足点がある場合、何をどの程度詳細に書けば、いずれの基準にもとづいて適切と思われるのか、ご教授いただければと思います。 ②コメント「事例が1つしかない」論文において本事例をもってのみ一般化することは難しく、SNS上の炎上現象に関する先行研究の結論を補完するものである、と記述したとおりです。 ③その他（細かい点）について第一のコメントは質的データ分析を行った際のコーディングについてのご質問かと思いますが、「女性が感情的である」というのはジェンダー・ステレオタイプ（「女は優しい」「男はたくましい」「女は世話好き」「男は数字ができる」のような、男女の性格や能力、役割、行動などに関する単純で固定化された考え方（森永 2013））のひとつで（Weigard et al. 2021）、こうした偏見が他者認知をゆがめ、性差別、なかでも善意的性差別（benevolent sexism）に繋がることは既存研究で明らかになっています（Glick & Fiske, 1996, 1997, 1999, Correll et al., 2020）日本語のオンライン環境においても、特に女性に対し「感情的である」として発話のパフォーマンスを攻撃することで、発言者の意見を抑え込むことが指摘されています（田中, 2020）。男性も「感情的になるな」と批判を受けることがあるとのご指摘ですが、これは「感情的になること」――前述のとおり、一般的に女性の性格や能力、役割、行動に紐付けられているステレオタイプですが――が誤っている、よくない点であると捉えられているため、指摘しあげつらわれていると考えられます。こうした理由から、「感情的である」というのは個人攻撃ではなく、女性差別的、すなわち対象者が属する（とみなされている）集団に対する差別的発言であると判断しました。他方、個人攻撃については、対象者が属する集団（この場合、女性という性グループや、メディア企業）ではなく、記者本人に対する攻撃をこのカテゴリーに分類しました。著作権とプライバシーの保護に留意しつつ、それと分かりやすい例を追加いたしました。なおコーディングについては、本稿ではデータを読みながら探索的に自由にコードを付す「生成的コーディング generative coding」を行いました。質的研究で採用されることの多いこのコーディングでは、解釈が重要な機能を果たし、主体的なものと考えられます。また、このため複数のコーディング作業者をおくことは稀で、この点は選定されたコード群からコードを選んで付し、複数のコーディング作業者を置いてその間の一致度を評価する「標準化コーディング standardized coding」とは異なることを申し添えます（大谷 2019）。オンラインハラスメント対策に関しては、先述のとおり、本稿は質的研究であり、実証や仮説検証を目的とするものではないこと、またResearch questionも「どうなっているか？」という記述的問いであり、「どうすればいいか？」という「処方的問い」ではないことから（大谷 2019）、本稿の分析から得られた知見を対策の一助とすることは回避しました。他方、主に英語圏の先行研究、及び「炎上」についての日本語の先行研究には対策案として採用されるべき貴重な知見も示されており、それらを日本語のインターネット環境や、個人・個人が所属する組織の実務的な観点から検討し、有用と思われるものを提言いたしました。こうした背景が分かりにくいことはご指摘のとおりと考えますので、加筆修正いたしました。小山ら（2019）の論文の文献リスト漏れについてご指摘ありがとうございました。その他の追加論文とともに挿入しました。参考文献 Correll SJ, Weisshaar KR, Wynn AT, et al.: Inside the Black Box of Organizational Life: The Gendered Language of Performance Assessment. Am. Sociol. Rev. 2020; 85(6): 1022–1050. https://doi.org/10.1177/0003122420962080 Glick P, Fiske ST: The Ambivalent Sexism Inventory: Differentiating Hostile and Benevolent Sexism. J. Pers. Soc. Psychol. 1996; 70: 491–512. https://doi.org/10.1037/0022-3514.70.3.491 Glick P, Fiske ST: Hostile and Benevolent Sexism. Psychol. Women Q. 1997; 21(1): 119–135. https://doi.org/10.1111/j.1471-6402.1997.tb00104.x Glick P, Fiske ST: The Ambivalence toward Men Inventory: Differentiating hostile and benevolent beliefs about men. Psychol. Women Q. 1999; 23(3): 519–536. https://doi.org/10.1111/j.1471-6402.1999.tb00379.x Weigard A, Loviska AM, Beltz A: Little evidence for sex or ovarian hormone influences on affective variability. Scientific Reports. 2021; 11, 20925. https://doi.org/10.1038/s41598-021-00143-7 大谷尚: 質的研究の考え方　研究方法論からSCATによる分析まで. 名古屋大学出版会; 2019. 田中東子: なぜ男性より女性のほうがインターネットで炎上しやすいのか　「女性らしくない女性を罰したい」.2020. (accessed 22 December 2021). Reference Source 小山耕平, 浅谷公威, 榊剛史, et al.:ネット炎上におけるユーザーの共振構造. The 33rd Annual Conference of the Japanese Society for Artificial Intelligence. 2019." } ] } ]	1	https://f1000research.com/articles/10-1164
https://f1000research.com/articles/11-138/v1	02 Feb 22	{ "type": "Research Article", "title": "Protein-Energy Nutritional Status of Moderately Low Protein Intake-Sago Diets Compared to Sufficiently Protein Intake-Rice Diets in Well-Nourished Lowlanders in Papua, Indonesia", "authors": [ "A. Yasmin Syauki", "Aki Ogawa", "Uli Rina Pelegia Simanjuntak", "Ingrid Gloria Mangiwa", "Miki Doi", "Suzumi Kageyama", "Rikako Inoue", "Nurpudji A. Taslim", "Yasuyuki Irie", "A. Yasmin Syauki", "Aki Ogawa", "Uli Rina Pelegia Simanjuntak", "Ingrid Gloria Mangiwa", "Miki Doi", "Suzumi Kageyama", "Rikako Inoue", "Nurpudji A. Taslim" ], "abstract": "Background: Protein inadequacy is prevalent in developing countries because of the high consumption of starchy staple foods. Sago, as a staple food in Papua Province, was eaten with less protein. This study aimed to analyze the nutritional status of protein-energy in well-nourished adults of the moderately low protein in-take (MLP)-sago group compared to the sufficient protein intake (SP)-rice group, in lowland Mimika, Papua. Methods: This cross-sectional-analytic study was conducted on 50 participants. Twenty-four-hour food recall, body composition, albumin, and complete blood count were used to assess the nutritional status. Results: There were no differences in the body compositions and albumin levels between the MLP-sago and SP-rice groups. Multivariate linear regression showed that the mean corpuscular volume (MCV) (β = -0.524, P = 0.007) was a predictive factor for albumin in the sago group, while in the rice group, hemoglobin (β = 0.354 P = 0.089) and white blood cell counts (β = 0.396, P = 0.059) were predictive factors. Conclusions: The MLP-sago and SP-rice groups exhibited no differences in the body and visceral protein; however different predictors of albumin were found be- tween the groups, suggesting an adaptive mechanism in the MLP-sago group to maintain normal albumin levels.", "keywords": [ "moderately low protein intake", "sago", "albumin", "MCV", "visceral fat", "basal metabolic rate", "well-nourished", "Papua." ], "content": "Introduction\n\nProtein is an essential element of the diet. It is required for the growth and maintenance of the 25,000 proteins that have been encoded by the human genome. The latest recommendation for protein requirements was released in 2007.1Access to quality nutrients, especially proteins, is a problem among poor populations in low-income countries. Countries in sub-Saharan Africa and South Asia have a prevalence of protein inadequacy of more than 5%. Proteins from animal-source foods in the food supplies of sub-Saharan Africa and Asia have been shown to be lower than the global average (20% and 32% vs. 40%, respectively). Most of the proteins (70%) consumed in sub-Saharan Africa are obtained from cereals and roots2 with diets being dominated by starchy staple foods, and nutrient-dense animal-source foods, fruits, and vegetables often being unavailable or unaffordable.3\n\nSago palm is a plant resource that has the most efficient carbohydrate-producing crop in the world. It produces starch and stores it in the trunk. The amount of production is between 150—300 kg of dry starch per plant or up to 25 tons of starch per hectare per year. It is approximately three to four times higher than that of rice, corn, or wheat, and 17 times higher than that of cassava. Sago palm is expected to provide a countermeasure for food insecurity resulting from the rapid population growth (95% being predicted in developing countries) and an increase in per capita food consumption. Sago can grow under natural conditions or can be cultivated in underutilized wetlands and peat swamps. However, the conversion of wetlands and peat swamps into industrial crop plantations has had a negative impact on the sustainability of sago palm production.4–6\n\nIndonesia has the largest area of sago palm plantations and sago forests in the world (estimated 85%) and the richest genetic diversity of sago palms. Sago palms in Indonesia, such as in Papua Island, Sulawesi Island, Maluku Island, Sumatra Island, Kalimantan Island, and Java Island, with the highest area being in Papua Island (estimated 95%). Papua was proposed as the center of sago palm diversity in Indonesia while Sulawesi and Kalimantan were the sources of diversity. The sago palms in Papua and West Papua grow in sago forests that have not been managed or cultivated, which can decrease the production.6\n\nBefore rice largely replaced these crops, sago together with taro and yam was claimed to be one of the oldest crops and former staple foods in large areas of Southeast Asia and Oceania. Papua Province has a huge sago forest of more than 1 million hectares in total. Coastal communities and the lowlanders in the Papua province consumed papeda (sticky dough, which is considered as cooked rice) and sago lempeng (roasted sago) as their food since ancient times and they still currently do so.6–9 Limited production and a higher price of sago compared to imported food products, subsidized particularly imported rice, resulting in a declining consumption of sago.6,8\n\nThe farming behavior of indigenous Papuan farmers is similar to “home-vegetable gardening”. They cultivated plants that are resistant to disease and have a minimum risk of failure, such as taro, sweet potatoes, cassavas, and bananas. Those commodities are also limited in quantities only for their consumption. For sago-eating people, wild sago was obtained in the forest and stored for one week’s consumption. Meanwhile, rice-eating people were not cultivating rice, as an alternate, they were provided rice from the government through “rice for the poor policy”. These behaviors were influenced by two perceptions that related to local wisdom in preserving nature. First, the potential of natural resources available in the ecological environment is a source to meet the food needs of the local population. Second, the potential of foods crops in nature is perceived as “a blessing” that should not be overused. This phenomenon affects the performance of Papuan farmers such as not cultivating agricultural land regularly, not developing market-oriented agricultural commodities, and tending to grow commodities that can be consumed in limited quantities.10\n\nRice policy was integrated into the national transmigration program in 1954/1955 and its implementation continued between the 1970s and the 1990s. This policy was continued by the central government through the “rice for poor” policy since the 2000s.11 Transmigration started in Papua in 1984 in the form of military transmigrants. Rice policy as part of the transmigration program also began and proceeded with the rice for poor policy since 2002.12–13 Native people who were eating sago as their staple food and changed to rice consumption were receiving rice for free from the government and maintained their home-vegetable garden of planting.10\n\nThe use of sago starch and sago-based food products spread in 21 of the 33 provinces of Indonesia as staple foods or snacks, especially in the coastal or lowland areas. Sago as local food has great potential to be developed to support food diversification because it contains high carbohydrate productivity. However, other nutrients, including proteins, are very low in sago. As a staple food, sago must be consumed together with a protein food source to fulfill the requirements of protein intake.7,9\n\nThe history of coastal communities with the consumption of sago as the main staple food shows that they are healthy, physically strong people, and reliable seafarers.7 Mimika is a low-lying district, and people consume sago as their staple food with less protein. Mimika is also classified as a district with a higher risk of food insecurity.14 However, to date, no report has implied protein deficiency symptoms in adults in this area, contrary to the strong muscular bodies of these people. Rice contains a certain level of protein and is consumed by local people. There is no data available on the health issues involving low protein intake among people who eat sago. This study aimed to analyze the protein-energy nutritional status of two adult populations with similar hereditary and environmental conditions, one who consumed sago with a moderately low protein intake, and the other, who consumed sago with sufficient protein intake.\n\n\nMethods\n\nThis cross-sectional-analytic study was a part of the main study on analysis of gut microbiota of the lowlanders in Mimika Regency, Papua, Indonesia, conducted in September 2019. The sample size of this study was based on the main study (50 participants with 25 in each group). The inclusion criteria were men and women lived in the lowland of Mimika Regency, Papua with ages ≥ 20 years, body mass indexes between 18,5—24,9 kg/m2, those who consumed traditional carbohydrate-based food (sago) with low protein intake (< 25 g/d) or who consumed modernized carbohydrate-based food with sufficient protein intake (≥ 25 g/d). Participants who had taken antibiotics within the preceding six months, who had taken laxatives, gastric motility medications, prebiotics, or probiotics containing foods or supplements within the preceding month, had a medical history of clinically significant diseases such as cancer, gastrointestinal disorders (irritable bowel syndrome, inflammatory bowel diseases, coeliac diseases, constipation, diarrhea, excessive bloating), autoimmune disorders, diabetes, heart diseases, renal failure or previous gastrointestinal surgery, infectious diseases, smokers or those with high alcohol consumption, were excluded from the study. The study was approved by Komite Etik Penelitian Kesehatan (Health Research Ethical Committee), of the Faculty of Medicine, Hasanuddin University, Makassar, Indonesia (Approval Recommendation Number: 554/UN4.6.4.5.31/PP36/2019 and protocol code: UH19070416). This study was also approved by the Ethics Committee of Okayama Prefectural University (Protocol Code Number: 19-55). Written informed consent was obtained from all participants involved in the study.\n\n2.1.1. Socio-demographics\n\nA questionnaire was used to collect information on the characteristics of the participants, such as socioeconomic- demographics (age, ethnicity, education, occupation, and income), medical histories (cancer, gastrointestinal disorders, autoimmune disorders, diabetes, heart diseases, renal failure, previous gastrointestinal surgeries, infectious diseases), consumption of medications or supplements (laxatives, gastric motility medications, and prebiotic or probiotic-containing foods or supplements), and lifestyles (smoking and alcohol consumption).\n\n2.1.2. Anthropometric Assessment\n\nParticipant height was measured using a mobile stadiometer (SECA 213). Body weight, body fat %, bone mass, total body water %, muscle mass, basal metabolic rate (BMR), and visceral fat were measured using a body composition monitor (TANITA BC 730). The measuring platform was placed on a hard, flat surface with minimal vibrations to ensure a safe and accurate measurement. Measurements were taken with minimal clothing and on an empty stomach (no meal before). The socks were removed, and the soles of the feet were cleaned before the participant stepped onto the measuring platform. Before measuring, personal data such as birth dates, gender, and height were input. The participant was asked to step onto the scale after the scale turned on, stand unassisted in the center of the platform and look straight ahead while standing relaxed but still. The participant was allowed minimal clothing, but a mobile phone, a wallet, and anything heavier were removed. After the measurements were taken, the readings were displayed automatically, including body weight, bone mass, total body water percentages, muscle mass, BMR, visceral fat, and body fat percentages. The assessment was performed twice, and the average was used for the analysis. One of the most common methods for measuring muscle strength is the isometric grip strength test. We measured the isometric grip strength using a handgrip dynamometer. The participant was asked to squeeze the dynamometer as hard as possible with each hand while in a standing position. Before beginning the grip strength testing procedures, the participant was asked a series of questions to obtain information about muscle conditions (any visible limitations for either hand, any surgery of his/her hand, any pain, aching or stiffness, right-handed, left-handed, or equally). The participant remained seated during the preparation and warm-up periods.\n\n2.1.3. Dietary assessment\n\nThe intake of energy, macronutrients (carbohydrate, protein, fat), micronutrients, and fiber were measured using basically 3-day non-consecutive days of a 24-hour food recall (only one person had two-day non-consecutive days). This assessment was performed on one or two weekdays and on one weekend day to obtain their usual dietary intakes. Face-to-face interviews were conducted to recall their food intake in the last 24-hour. On estimating portion sizes of the food, plastic food models were used with information on portion sizes, for example, food model of standardized rice with portion sizes information or food model of standardized fish with portion sizes information. Common household measures such as household cups, bowls, spoons, rice spoons, and food photographs were also used to assist the individuals in estimating their portion of the food consumed. Observations on the fish seller were conducted to have accurate data on the type and the portion size of fish. Cooking methods of their local staple food have also been observed to have a precise recipe. At the beginning of the study, a meeting was held together with the head of the public health center to explain the purpose, the benefits, the measurements, and the team member of the study to establish communication with our study population. On collecting food intake data, we were accompanied by staff from the public health center, who is familiar with our study population. The dietary intake was analyzed using the Nutrisurvey 2007 application (www.nutrisurvey.de).\n\n2.1.4. Blood Test\n\nThe complete blood counts and plasma albumin levels were measured by Prodia Laboratory, Jayapura, Papua, Indonesia.\n\nAll the data were expressed as means ± SDs and medians (minimum, maximum). The data normality distribution was determined using the Shapiro-Wilk test. The differences between groups were determined by the independent t-test (normally distributed data) or Mann-Whitney test (non-normally distributed data). The Pearson test was used to evaluate the correlation between albumin levels and other parameters because albumin was normally distributed. Parameters that correlated with albumin (P < 0.25) were further included in multivariate linear regression analyses. Multivariate regression tests were performed on both the rice and sago groups. The results of the regression models are shown as B, standard error, β, t, and p values. The Pearson’s test was also used to evaluate the correlation of the laboratory profiles, anthropometry profiles, and intake profiles of both the rice and sago groups. All the statistical analyses were performed using the Statistical Package for the Social Sciences version 27 (SPSS Inc, Chicago, IL, USA). Statistical significance was established at a P-value < 0.05.\n\n\nResults\n\nFifty participants were recruited with 25 each with 12 male and 13 female in the rice and 11 male and 14 female in sago groups. Participants in the sago group were significantly older than those in the rice group (52.91 vs. 43.28-year, P < 0.05). Most of the participants in both groups were of Kamoro ethnicity (Table 1). The educational background of both groups was predominantly 6—9 years of schooling. However, > 9 years of schooling was the second most common (24%) educational background in the rice group. Farming with a home-vegetable garden was the most common occupation in both groups; however, temporary employment was the second most common occupation in the rice group. Most of the participants in both groups had incomes < 1.000.000 IDR per month; however, in the rice group, 8% had an income > 2.000.000 IDR per month.\n\n* Significantly different (P < 0.05) by Mann-Whitney U test with the rice group\n\nThe intake of most of the nutrients between the rice and sago groups was significantly different. Protein intake in the sago group was significantly lower than in the rice group (19.9 g/d vs. 36.7 g/d, P < 0.001). However, the carbohydrate and fiber intake were higher in the sago group than in the rice group (245.5g/d vs. 171.7 g/d, P < 0.001; 5.0 g/d vs 3.3 g/d, P = 0.001, respectively). The micronutrient intake profile of the rice group was significantly higher than that of the sago group (Table 2).\n\na Significant difference between the rice and sago groups with the independent t-test;\n\nb Significant difference between the rice and sago groups with Mann-Whitney U test.\n\nOur food groups showed that the rice group gained the largest energy from the rice (607 kcal/day), while the sago group did it from sago (810 kcal/day). Rice (11.0 g/day) and fish (13.1 g/day) were the sources of protein in the rice-eating group, while in the sago group, fish (12.2 g/day) was the major source of the protein (Table 3 and 4).\n\nBody composition (muscle mass, fat mass, visceral fat, and basal metabolic rate) showed no significant differences between the rice and sago groups. The hand-grip strength did not differ between the groups (Table 5). Muscle strength of both groups showed within in normal range. In the rice group, men had a median of 37.3 kg and women had a mean of 22.3 kg with the reference value in men 35.5—55.3 kg and women 18.9—32.7 kg for 40—44-year-old. While in the sago group men had a median of 36.7 kg and women had a median of 22.5 kg with the reference value in men 34.7—54.5 kg for 45—49-year-old and women 17.7—31.5 kg for 55—59-year-old.\n\nLaboratory profiles showed a significant difference between the rice and sago groups in the hemoglobin and hematocrit levels of the males (13.7 g/dL vs 12.6 g/dL, P = 0.044; 43% vs 41% P = 0.027, respectively). Men in the rice group had normal hemoglobin levels, while those in the sago group had hemoglobin levels lower than the reference values. Meanwhile, the hematocrit levels of the men in both groups were within the reference values. The hemoglobin levels in the women in both groups were lower than the reference values but did not differ between the groups. A lower MCV value than the reference value was found for both rice and sago groups. Iron intake did not differ between groups of men and women but was lower than the reference value Serum albumin levels did not differ between the rice and sago groups and were within the reference values (Table 6).\n\n* Significantly different between the rice and sago groups with Mann- Whitney U test.\n\nA multivariate analysis of albumin for both groups was conducted to analyze the correlation of body composition and laboratory profile with albumin. Multivariate linear regression analysis showed that a better prediction of albumin in the rice group was featured by hemoglobin (β = 0.354, P = 0.089), and the white blood cell counts (β = 0.396, P = 0.059). This model explained 12.8% of the albumin variability (Table 7). We accepted this model even though P-value > 0.05 (the software was set to maintain variable at least P-value = 0.1) because hemoglobin and albumin were found to be correlated in anemic patients. While albumin and white blood cell were reported to be correlated in diabetic patients.\n\nOn the one hand, in the sago group, the better model for the multivariate association in determining albumin was characterized by the MCV (β= -0.524, P = 0.007). This model explained 24.3% of albumin variability in the sago group (Table 8).\n\nIn the sago group. there were significant correlations with the MCV and visceral fat (r = 0.415, P = 0.039), the visceral fat and basal metabolic rates (r = 507, P = 0.010).\n\n\nDiscussion\n\nThe present study showed that there were no differences in the body composition and serum albumin levels between the sago-moderately low protein and rice-sufficient protein groups. The hemoglobin and hematocrit levels in the males were the only items that were significantly different between the two groups. This study also analyzed the serum albumin level as a marker for protein-energy malnutrition caused by insufficient protein intake. The multivariate analysis revealed that the predictive factors of serum albumin levels were different between the two groups. Hemoglobin and white blood cell (WBC) counts were the determinants of the serum albumin level in the rice-sufficient protein group. On the other hand, the mean corpuscular volume (MCV) was a predictor of the serum albumin levels in the sago-moderately low protein group.\n\nThe sago-eating participants were older than the rice-eating participants. A similar finding was reported in a study in Riau Province, Indonesia, where the people who consumed more sago were older (> 50 years old) compared to the those who consumed less sago.15 Sago is considered to be the major food of ancient times in the sago-producing areas of Indonesia. Recently sago consumption has been reduced and replaced by the consumption of rice. The socioeconomic status of the rice consumers in our study showed that they had higher educational backgrounds, occupations, and incomes than the sago consumers. A study in Maluku, Indonesia also showed that better household incomes and education will reduce sago consumption and production, shifting to rice consumption. Rural people also perceived sago as inferior food, while rice perceived as superior food8,.11\n\nMost of the nutrient intakes of sago-eating participants such as protein and micro-nutrient intake were lower than that those of rice-eating participants in our study. Energy intakes, carbohydrate intakes, and fiber intakes were higher in the sago group than in the rice group. Syartiwidya et al had similar findings that participants consumed more sago (> 140 g/day) compared to participants who consumed less sago (< 140 g/day) and had higher percentages of severe deficits in the adequacy of protein levels (43% vs. 38.3%).15 Based on the Indonesian Nutrient Composition Food, while sago per 100 g edible portion had a higher carbohydrate content than white rice cooked (85.6 g vs. 26.0 g) it had a lower protein content (0.6 g vs. 2.4 g).16\n\nThis is the first study on the dietary intake of lowlanders in Papua, Indonesia with energy and protein intake were 1029 kcal and 36.7 g for the rice group and 1233 kcal and 19.9 g for the sago group. A study by Okuda, T et al in 1981 on Papua New Guinea highlanders showed that the mean daily energy intake was 2390 kcal, and the daily protein intake was 35.2 g. This profile was exceptionally high because there was a yearly festival season in the village when the data were collected. In comparison with them, our result on lowlanders in Papua, Indonesia had lower energy intake. The lowlanders in Papua, Indonesia were having daily foods with small varieties and in contrast, the highlanders in Papua New Guinea were having special food for the yearly festival. The yearly festival season on highlanders and the wages from coffee plantations can affect these differences.17\n\nThe dietary intake of the two groups showed that the main differences were in the source of carbohydrate and protein intake. Resistant starch type 3 (RS 3) derived from sago contained higher RS (31—38%) than those derived from rice starch (21—26%). This implicated on the production of short-chain fatty acid (SCFA) mainly butyrate as the preferred energy source for colonocytes.18\n\nStunting was a nutritional problem in Indonesia. According to Indonesian National Health and Research Survey in 2018, national data showed there was a decreasing prevalence of severe stunting from 18.0% in 2013 to 11.5% in 2018 but slightly increased stunting from 19.2% in 2013 to 19.3% in 2018.19 Mimika district had better stature compared to other districts in Papua province (Mimika profile is 5.68 percentage of severe stunting, 14.29% of stunting, and 80.03% of normal).20 A study by Fikawati, S. et al in 2021 among preschool children in Central Jakarta, Indonesia found that children who lacked energy and protein intakes were at a higher risk of stunting than children who had sufficient intakes. Macronutrient intakes are important and should be consumed in sufficient quantities every day to prevent stunting.21 Inadequate energy and protein intake in our population study can be the risk of having stunting on their children. Height among our participants showed that their height was comparable to the highlanders of Papua New Guinea. Highlanders of Papua New Guinea had a height of 158.3 cm, and it was similar to our height lowlanders of Papua Indonesia 158.5 cm for the rice group and 160.5 cm for the sago group.17 The sago-eating people can get more resistant starch 3 compared to rice-eating people. This implicated to have a higher production of short-chain fatty acid (SCFA) mainly butyrate, the energy source for colonocytes in the sago-eating people than rice-eating people.18 Meanwhile, the inadequate protein should be corrected to the level adequate to prevent stunting in children in their family members.\n\nThe amount of protein intake in the sago and rice groups was 0.35 g/kg/d and 0.66 g/kg/d, respectively. The minimum intakes for nitrogen equilibrium were the main factors of the lower limits of successful adaptation at which an appropriate body composition can be maintained. The mean adult requirement value of 0.66 g/kg indicated by the nitrogen balance studies implied an overall efficiency of utilization of dietary proteins of approximately 50% in replacing the obligatory nitrogen loss. The risk of protein deficiency started to increase when the intake decreased to below the range of the true minimum requirements (that is, a value that is currently unknown but likely to be between 0.40 and 0.50 g/kg/d).22 A series of studies of highlanders in Papua New Guinea, who depended largely on sweet potatoes and vegetables with low protein intake (4% of total calories) showed negative nitrogen balances with little malnutrition within the population. It was hypothesized that there was a fixation of nitrogen by the intestinal microflora.23 A study by Huang et al suggested that there was no intestinal nitrogen fixation to occur in sweet potato eaters in slightly below requirement levels of protein intake.24 Intake of protein also found significant differences at different locations on the highlanders of Papua New Guinea using a delta nitrogen stable isotope (15N), the discriminant factor between diets and scalp hair. The estimated 15N values were correlated negatively with several indicators of animal protein intake.25 A study developing and validating a semi-quantitative food frequency questionnaire (FFQ) for assessing protein intake found that the highlanders of Papua New Guinea did not meet the biologically required protein intake. Although closer to an urban center, it showed higher protein intake than the more remote communities.26\n\nSerum albumin levels in the sago group did not differ from those in the rice group and fell within the normal range. Multivariate linear regression analysis showed different predictive factors for albumin levels in these groups. Hemoglobin and white blood cells were factors related to albumin in the rice group, while MCV was a predictor of albumin in the sago group. Based on our working hypotheses, basal metabolic rate influences serum albumin levels in the sago group indirectly. Protein intake in the sago group can be classified as a moderately low protein intake. A moderately low protein intake does not change the basal metabolic rate or energy expenditure. Serotonergic and β -adrenergic systems are believed to be involved in the mechanism of the changes in energy balance. These are only two of the neurotransmitter systems involved in regulating food intake. Moderately low protein-high carbohydrate increases adiposity and fat in the liver. In an animal study, the lowest protein consumption increased the proportion of energy deposited as carcass fat. The higher the fat in the liver, the lower is the iron in the liver tissue. In this study, lower iron levels were marked by a lower level of MCV. This condition induces an adaptation mechanism of protein metabolism to maintain body protein balance (albumin within normal range). We assumed that there was decreased protein turnover (protein synthesis, amino acid oxidation, protein degradation) with maintained post-absorptive whole-body protein and basal muscle protein synthesis as the mechanism of long-term of low proteins intakes in our participants (Figure 1).\n\nAbbreviations: MCV mean corpuscular volume; g gram; dL deciliter; fl femtoliter; kcal kilocalories; kg kilogram, d day.\n\nThe serum albumin levels in the moderately low protein sago diet showed a negative prediction according to the MCVs. Similar findings were obtained in a study by Suk-Hwan Yang on the relationship between liver function tests and MCVs in 157 persons (patients with liver disease and a healthy control group) indicating that albumin was related significantly and negatively with MCVs (stepwise multiple regression analysis). The MCV levels were higher, but albumin levels were lower in patients with liver disease than in the controls.27 The adaptation mechanism of albumin in the present study was triggered by a decrease in the MCV levels (iron serum). An in vitro study by Higashida et al found that iron deficiency decreases iron-containing protein and reduces protein synthesis in basal and BCAA- and insulin-stimulated conditions in muscle cells.28 Animal and human studies have shown that there is a reduction in the synthetic and catabolic rates of albumin in protein-deficient states. There was an increasing transfer of albumin from the extravascular pool to the intravascular pool to maintain the serum albumin within normal levels.29,30 Animal studies have also shown a decrease in albumin synthesis (as a percentage of total liver protein synthesis) from 15% to 8% on a protein-free diet (2-9 days) in rats.31 Reducing the protein intake of humans from required intake (0.6 g/kg/d) to inadequate level (0.1 g/kg/d) will decrease leucine flux, body protein synthesis, and protein breakdown with a smaller reduction in leucine oxidation.32\n\nThe rate of albumin synthesis in healthy participants consuming a diet with very low protein (e.g., 10 g/day) in an isocaloric diet, showed a decrease in albumin synthesis by 20%—65%. However, when both protein and calories are restricted (i.e., starvation), albumin synthesis remained close to normal. The catabolism of body proteins to provide energy, released sufficient amino acids to maintain normal albumin synthesis.33 Albumin synthesis was also modulated by the proportion of animal and vegetable protein in the diet. Studies on isoenergetic and isonitrogenous diets in healthy men showed that the albumin synthesis rate in the group consuming 63% of vegetable protein was reduced in comparison with the group consuming 74% of animal protein.34 Albumin is the most abundant antioxidant in whole blood, and oxidative stress has now emerged as a major pathway with pathological relevance for many cardiovascular diseases, such as atherosclerosis, coronary artery diseases, heart failure, atrial fibrillation, strokes, and venous thromboembolism.35 A study on the very old, centenarian, and supercentenarian in Japan showed that plasma albumin levels were almost associated with all-cause mortality in these populations. Plasma albumin levels were correlated significantly with cholinesterase levels, inflammation, and NT-pro BNP levels (biomarkers of endogenous cardioprotective molecules).36\n\nMuscle mass in the moderately low protein sago diet was not significantly different from that in the sufficient protein rice diet. Healthy humans can maintain lean body mass and body protein balance when protein intake is restricted. Integrated and adaptive metabolic changes in the body occur by decreasing amino acid oxidation and protein degradation with a more efficient use of amino acids derived from protein degradation. Maintaining skeletal muscle protein synthesis is an important component of the “adaptation” to low protein intake.37 A randomized parallel study by Hursel et al, with 15 participants either high (2.4 g/kg/d) or low protein intake (0.4 g/kg/d) showed that low protein intake induces prolonged adaptation of body mass and fat-free mass by lowering body protein turnover rates (protein synthesis, protein breakdown, and protein oxidation), but maintains post-absorptive whole body net protein balance and maintains basal muscle protein synthesis in 12 weeks.38 Mosoni et al showed that short-term food deprivation induced the inhibition of muscle protein synthesis and liver protein synthesis after 112—114 hours of food deprivation and 5—7 hours of re-feeding in rats. After re-feeding, liver synthesis was more stimulated than muscle protein synthesis. A coordinated response of liver and muscle protein metabolism allowed sparing of muscle proteins during food deprivation at the expense of liver proteins.39\n\nVisceral fat and MCV were correlated positively in the moderately low protein sago group. Different findings from an animal study by Visscher et al (2017) showed that the level of fatty acids in the liver had a strong negative correlation with the iron content. The livers of turkeys that died from hepatic lipidosis were analyzed for their fat and iron levels. The higher the fat content in the liver, the lower the iron content in the liver tissue.40 A study by Siddique et al, on nonalcoholic fatty liver disease patients, found that iron deficiency was prevalent, and this was associated with the female sex, increased body mass indexes, and non-white race. Interestingly, serum hepcidin levels were low in iron-deficient participants, indicating that serum hepcidin was not a primary cause of iron deficiency, rather it was a physiological response to decreasing levels of iron.41 Hepatic iron can cause liver injury. Adipose tissue iron can be linked to adipose tissue function, including the dysregulation of adipokines, enhanced adipose tissue lipolysis, and adipose tissue inflammation. These might be the possible mechanisms linking adipose tissue iron to liver injury.42 Unfortunately, serum iron and other iron profiles were not assessed in our study, but low MCV was related to iron deficiency. Low MCV was found in both groups, and iron intake was below the requirement. We assumed that the iron levels were related to the visceral fat in the sago-moderately low protein group but not in the rice-sufficient protein group.\n\nIn our study, sago-eating participants with a moderately low protein diet showed more visceral fat (liver fat) than rice-eating participants. A study by Du et al, on food intake, energy balance, and serum leptin concentrations in rats fed low-protein diets found several low levels of dietary proteins from total calories (2%, 5%, 8%, 10%, 15%, and 20% casein) influenced their energy intake and body fat. The lowest protein consumption increased the proportion of energy deposited as carcass fat. The body fat content showed a positive correlation with serum leptin concentrations.43 Another animal study by Pezeshki et al, showed that diets with 10% moderately low protein calories in obesity-prone rats had an increased liver fat percentage (hepatic lipidosis). While this moderately low protein diet caused hyperphagia (increasing energy intake) without altering energy expenditure, body fat, and lean mass, it promoted hepatic lipidosis.44\n\nThe sago group with moderately low protein intake showed a similar basal metabolic rate compared to the rice group. A study by Pezeskhi et al, on the effects of diets varying in protein concentrations on energy balance in obesity-prone rats found that protein-free (0% protein calories) diets decreased energy intake and increased energy expenditure, very low protein (5% protein) diets increased energy intake and expenditure, whereas moderately low protein (10% protein) diets increased energy intake without altering expenditure, relative to the control diet (15% protein). Serotonergic and β-adrenergic signaling coupled with the upregulation of key thermogenic markers in brown fat and skeletal muscle were thought to be the mechanism of the change in energy balance.44 Different findings by Miyatani et al on the basal metabolism of various types of protein diets with the rice diet and sweet potato diet of the Papua New Guinea highlanders found that there were no significant differences in the basal metabolic rates (BMRs) among the sweet potato diet with low protein (0.3 g/kg/d), the sweet potato diet with protein (0.5 g/kg/d), rice diet with low protein (0.6 g/kg/d), and rice diet with protein (1.4 g/kg/d), however, the respiratory quotients were different.45\n\nOur study found that body composition and serum albumin levels were not significantly different between the groups of lowlanders of Mimika, Papua, Indonesia. Multivariate linear regression showed that there were different predictors of albumin between the sago-eating group and the rice-eating group. An adaptation mechanism for the sago-moderately low protein intake may have maintained the albumin levels within the normal range.\n\nThis study was a part of the main study on analysis of gut microbiota of rice-eating people with sufficient protein intake and sago-eating people with low protein intake of the lowlanders in Mimika Regency, Papua, Indonesia. We are interested in their habitual low protein intake of sago-eating people, but they appeared to be healthy. Therefore, the measurement of protein intake was carried out carefully. Besides using plastic fish models with the portion size, we also looked at the fish that they consumed in the fish seller and compare them with our plastic models. On the other hand, on the measurement of carbohydrates, we used plastic rice models with the standard portion size such as “100 grams of rice on a plate”. This standard portion sizes of plastic models may not capture their real carbohydrate intake precisely. They might have eaten more than they reported. Underreporting energy intakes were prevalent in economically deprived populations, and this is one of our limitations in this study. Therefore, we think that underreporting of energy intake is from underreporting of carbohydrate intake. A review article by Maurer, J. et al, 2006, on the psychosocial and behavioral characteristics related to energy misreporting explained that lower carbohydrate intake was one of the commonest dietary patterns of underreporting.46 Underreporting of energy intakes in our study also can be influenced by their education level and their social-economic status. A study by Olendzki, B.C., et. al in 2008 found that energy intake underreporting was prevalent in the low-income, low literacy of the Carribean Latino population.47 Other factors associated with misreporting energy intake were demographics and diet. Women, older adults, and people with less education tend to underreport energy intake.46 A study by Sawaya, A.L. et, al in 1996 on comparing four different methods of the dietary survey such as a 7 day-weighing method, a 24-hour food recall (in duplicate), and two types of food intake frequency survey among young and older women with doubly labeled water (DLW) found that the total energy intake (TEI) by the 24-hour food recall method was the closest to the total energy consumption calculated by DLW for young women. The TEI/DLW was 86.7 percentage for young women (mean age 25.2 ± 3.5 years) and for older women (74.0 ± 4.4 years) was 75.2 percentages.48\n\nAnother limitation in our study was the limited number of indicators for assessing the energy-protein nutritional status such as urinary excretion of nitrogen per unit of creatinine to measure nitrogen losses. No data on menopause in relation to iron metabolism (there were eleven women with the age 55 to 64 years old and it was the age of menopause period according to WHO) were obtained. The other limitation of the study was the mismatch in some parameters of the two groups (the sago group was older than the rice group, some participants in the rice group had better socioeconomic status). Further research is needed to examine the interactions between the liver (fatty acid, iron, and albumin profiles) and the muscles (amino acid profile) with the regulatory hormones in participants with prolonged sago-moderately low protein intakes.\n\nInvestigating the adaptation mechanisms of protein metabolism to maintain albumin levels within the normal range may have a beneficial impact on gerontology. Malnutrition, with hypoalbuminemia as an indicator, among the aged population is an important health care problem, as this problem is prevalent across the world, especially among the elderly aged 75 years or older. Malnutrition was not confined to institutionalized or hospitalized elderly individuals but was also seen in community-dwelling genarians. The result of a 5-year and 7-year longitudinal study in Japan showed that decreasing albumin level was associated significantly with aging among community-dwelling older adults aged 65 and over.49,50 An epidemiological survey found that lower albumin levels, even within the normal range, were related factors of frailty measures, trace elements, and inflammation markers in the general population.51\n\n\nConclusions\n\nProtein inadequacy was prevalent in developing countries including Indonesia, because their diet comprises mainly starchy staples. Our study on local people found no differences in the body compositions and albumin levels in the sago- moderately low protein intake group with the rice-sufficient protein intake group. Albumin, as an indicator of long-term adaptation, had different determinant factors in both groups. Albumin in a moderately low protein intake-sagodiet was predicted negatively by MCV, while in a sufficient protein intake-rice diet, it was predicted positively by the hemoglobin and WBC. The mechanism of adaptation to albumin in a moderately low-protein diet was induced by low levels of MCV (iron deficiency). Decreasing protein turnover (protein synthesis, amino acid oxidation, protein degradation) and the maintenance of post-absorptive whole-body protein and basal muscle protein synthesis preserved albumin levels within the normal range. This mechanism can be beneficial for the elderly (as an increase in the population around the world) who have low albumin levels. Further studies are needed to examine the interactions between the liver (fatty acid, iron, and albumin profile) and muscle (amino acid profile) with the regulatory hormones for the long-term adaptations of a moderately low protein intake- sago diet.\n\n\nData availability\n\nFigshare: Data Protein-Energy Nutritional Status of Lowlanders Mimika, Papua, Indonesia\n\nhttps://doi.org/10.6084/m9.figshare.17104568.v3\n\nThe project contains the following underlying data:\n\nRaw data of socio-economic, nutrient intake, food groups, body composition, and laboratory profile.\n\nData are available under the terms of the Creative Common Zero “No rights reserved” data waiver (CCO1.0 Public domain dedication).\n\nFigshare: Example menu of moderately low protein intake-sago diet and sufficient protein intake-rice diet of lowlanders Mimika, Papua, Indonesia.\n\nhttps://doi.org/10.6084/m9.figshare.17111075.v3\n\nThe project contains the following underlying data:\n\nExample menu of the sago diet and the rice diet in a day. Data are available under the terms of the Creative Common Zero “No rights reserved” data waiver (CCO1.0 Public domain dedication).\n\nFigshare: Iron intake of moderately low protein intake-sago diet and sufficient protein intake-rice diet of lowlanders Mimika, Papua, Indonesia based on sex.\n\nhttps://doi.org/10.6084/m9.figshare.18394394.v3\n\nThe project contains the following underlying data:\n\nIron intake profile of the sago diet and the rice diet based on sex.\n\nData are available under the terms of the Creative Common Zero “No rights reserved” data waiver (CCO1.0 Public domain dedication).\n\nFigshare: Correlation between MCV level, visceral fat, and basal metabolic rate of moderately low protein intake-sago diet of lowlanders Mimika, Papua, Indonesia.\n\nhttps://doi.org/10.6084/m9.figshare.18394433.v3\n\nThe project contains the following underlying data:\n\nCorrelation test between MCV level, visceral fat, and basal metabolic rate of moderately low protein intake-sago diet.\n\nData are available under the terms of the Creative Common Zero “No rights reserved” data waiver (CCO1.0 Public domain dedication).\n\nFigshare: STROBE Guideline Checklist of Cross-Sectional Study of Protein-Energy Nutritional Status of Lowlanders, Mimika, Papua, Indonesia. https://doi.org/10.6084/m9.figshare.17109269.v3\n\nThe project contains the checklist of STROBE guidelines of observational studies (cross-sectional study of protein-energy nutritional status of lowlanders, Mimika, Papua, Indonesia).\n\nData are available under the terms of the Creative Common Zero “No rights reserved” data waiver (CCO1.0 Public domain dedication).", "appendix": "References\n\nWorld Health Organization: Protein and Amino Acid Requirements in Human Nutrition. Report of a joint WHO/FAO/UNU Expert Consultation Geneva, Switzerland: WHO Press; 2007; 1–34.\n\nGhosh S, Suri D, Uauy R: Assessment of protein adequacy in developing countries: quality matters. Food Nutr. Bull. 2013; 34: 244–246. PubMed Abstract \| Publisher Full Text\n\nRuel MT, Haris J, Cunningham K: Diet Quality in Developing Countries. In Diet Quality: An Evidence-Based Approach. Preedy VR, et al., editors. New York, USA: Springer Science+Business Media; 2013; Volume 2. , pp. 239–260.\n\nHiroshi E: Potency of Sago Palm as Carbohydrate Resource for Strengthening Food Security Program. J. Agron. Indonesia. 2009; 37: 209–218.\n\nKarim AA, Tie AP-L, Manan DMA, et al.: Starch from the Sago (Metroxylon sagu) Palm Tree—Properties, Pro-spects, and Challenges as a New Industrial Source for Food and Other Uses. Compr. Rev. Food Sci. Food Saf. 2008; 7: 215–228. PubMed Abstract \| Publisher Full Text\n\nHiroshi E, Tokada Y, Johnson, et al.: Sago Palm Multiple Contributions to Food Security and Sustainable Livelihoods. Singapore, Singapore: Springer Nature; 2018; 10–62.\n\nBantacut TS: Sumberdaya untuk Penganekaragaman Pangan Pokok. Pangan. 2011; 20: 27–40.\n\nGirsang W: Sago Food Product Development for Food Security in Small Islands, Maluku, Indonesia. Int. J. Sci. Eng. Res. 2017; 8: 704–712. Publisher Full Text\n\nMetaragakusuma A, Katsuya O, Bai H: An Overview of The Traditional Use of Sago for Sago-based Food Industry in Indonesia. KnE Life Sciences. 2016; 3: 119–124. Publisher Full Text\n\nTurua U, Hadi S, Juanda B, et al.: Ekologi dan Budaya Petani Asli Papua dalam Usaha Tani di Kabupaten Keerom. Sosiohumiora. 2014; 16: 234–241. Publisher Full Text\n\nGirsang W: Socio-Economic Factors That Have Influenced the Decline of Sago Consumption in Small Islands: A Case in Rural Maluku, Indonesia. South Pacific Studies. 2014; 34: 99–116.\n\nFearnside P: Transmigration in Indonesia: Lessons from Its Environmental and Social Impacts. Environ. Manag. 1997; 21: 553–570. Publisher Full Text\n\nRice Policy Analysis in Indonesia: Then and Now.Reference Source (Accessed on 3 August 2021).\n\nMinistry of Agriculture’s Food Security Agency of Indonesia (Badan Ketahanan Pangan Kementerian Pertanian): Indeks Ketahanan Pangan Indonesia (Indonesian Food Security Index). Jakarta, Indonesia: Ministry of Agriculture of Indonesia; 2018; 8–13.\n\nSyartiwidya, Martianto D, Sulaeman A, et al.: Preference for Sago and Nutrient Intake among Communities Consuming Sago in Kepulauan Meranti District, Riau Province, Indonesia. J. Gizi Pangan. 2019; 14: 91–98. Publisher Full Text\n\nDirectorate General of Public Health Directorate of Public Nutrition of Indonesia (Direktorat Jenderal Kesehatan Masyarakat. Direktorat Gizi Masyarakat): Tabel Komposisi Pangan Indonesia (Indonesian Food Composition Table). Jakarta, Indonesia: Ministry of Health of Indonesia; 2018; 9–15.\n\nOkuda T, Kajiwara N, Date C, et al.: Nutritional Status of Papua New Guinea Highlanders. J. Nutr.Sci.Vitaminol. 1981; 27: 319–331. PubMed Abstract \| Publisher Full Text\n\nPurwani EP, Purwadaria T, Suhartono MT: Fermentation RS3 derived from sago and rice starch with Clostridium butyri-cum BCC B2571 or Eubacterium rectale DSM 17629. Anaerobe. 2012; 18: 55–61. Publisher Full Text\n\nResearch and Health Development Unit of Ministriy of Heatlh of Indonesia (Badan Penelitian dan Pengembangan Kesehatan): Laporan Nasional RISKESDAS 2018 (Report of National Research and Basic Health 2018). Jakarta, Indonesia: Ministry of Health of Indonesia; 2018; 562.\n\nResearch and Health Development Unit of Ministriy of Heatlh of Indonesia (Badan Penelitian dan Pengembangan Kesehatan): Laporan Provinsi Papua RISKESDAS 2018 (Report of Papua Province Research and Basic Health 2018). Jakarta, Indonesia: Ministry of Health of Indonesia; 2018; 411.\n\nFikawati S, Syafiq A, Ririyanti R, et al.: Energy and protein intakes are associated with stunting among preschool children in Central Jakarta, Indonesia: a case-control study. Malays. J. Nutr. 2021; 27: 081–091. Publisher Full Text\n\nMillward DJ: An adaptive metabolic demand model for protein and amino acid requirements. Br. J. Nutr. 2003; 90: 249–260. PubMed Abstract \| Publisher Full Text\n\nYoung VR, Marchini JS: Mechanisms and nutritional significance of metabolic responses to altered intakes of protein and amino acids, with reference to nutritional adaptation in humans. Am. J. Clin. Nutr. 1990; 51: 270–289. Publisher Full Text\n\nHuang PC, Lee NY, Chen SH: Evidences suggestive of no intestinal nitrogen fixation for improving protein nutrition status in sweet potato eaters. Am. J. Clin. Nutr. 1979; 32: 1741–1750. Publisher Full Text\n\nNaito YI, Morita A, Natsuhara K, et al.: Association of protein intakes and variation of diet-scalp hair nitrogen isotopic discrimination factor in Papua New Guinea highlanders. Am. J. Phys. Anthropol. 2015; 158: 359–370.\n\nMorita A, Natsuhara K, Tomitsuka E, et al.: Development, validation, and use of a semi-quantitative food frequency questionnaire for assessing protein intake in Papua New Guinean Highlanders. Am. J. Hum. Biol. 2015; 27: 349–357. PubMed Abstract \| Publisher Full Text\n\nYang S-H: Relationship Between Mean Corpuscular Volume and Liver Function Test. Korean J Clin Lab Sci. 1996; 28: 134–139.\n\nHigashida K, Inoue S, Nakai N: Iron deficiency attenuates protein synthesis stimulated by branched-chain amino acids and insulin in myotubes. Biochem. Biophys. Res. Commun. 2020; 531: 112–117. Publisher Full Text\n\nHoffenberg R, Black E, Brock JF: Albumin and gamma-globulin tracer studies in protein depletion states. J. Clin. Invest. 1966; 45: 143–152. PubMed Abstract \| Publisher Full Text\n\nJames WPT, Hay AM: Albumin metabolism: effect of the nutritional state and the dietary protein intake. J. Clin. Invest. 1968; 47: 1958–1972. PubMed Abstract \| Publisher Full Text\n\nPain VM, Clemens MJ, Garlick PJ: The effect of dietary protein deficiency on albumin synthesis and on the concentration of active albumin messenger ribonucleic acid in rat liver. Biochem. J. 1978; 172: 129–135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMotil KJ, Matthews DE, Bier DM, et al.: Whole-body leucine and lysine metabolism: response to dietary protein intake in young men. Am. J. Phys. 1981; 240: 712–721.\n\nLevitt DG, Levitt MD: Human serum albumin homeostasis: a new look at the roles of synthesis, catabolism, renal and gastrointestinal excretion, and the clinical value of serum albumin measurements. Int J Gen Med. 2016; Volume 9: 229–255. PubMed Abstract \| Publisher Full Text\n\nCaso G, Scalfi L, Marra M, et al.: Albumin synthesis is diminished in men consuming a predominantly vegetarian diet. J. Nutr. 2000; 130: 528–533. PubMed Abstract \| Publisher Full Text\n\nArques S: Serum albumin and cardiovascular disease: State-of-the-art review. Ann. Cardiol. Angeiol. 2020; 69: 192–200. Publisher Full Text\n\nHirata T, Arai Y, Yuasa S, et al.: Associations of cardiovascular biomarkers and plasma albumin with exceptional survival to the highest ages. Nat. Commun. 2020; 11: 3820. PubMed Abstract \| Publisher Full Text\n\nGaribotto G, Picciotto D, Saio M, et al.: Muscle protein turnover and low-protein diets in patients with chronic kidney disease. Nephrol. Dial. Transplant. 2020; 35: 741–751. PubMed Abstract \| Publisher Full Text\n\nHursel R, Martens EA, Gonnissen HK, et al.: Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy. PLoS One. 2015; 10: e0137183. PubMed Abstract \| Publisher Full Text\n\nMosoni L, Malmezat T, Valluy M-C, et al.: Muscle and Liver Protein Synthesis Adapt Efficiently to Food Deprivation and Refeeding in 12-Month-Old Rats. J. Nutr. 1996; 126: 516–522. PubMed Abstract \| Publisher Full Text\n\nVisscher C, Middendorf L, Günther R, et al.: Fat content, fatty acid pattern and iron content in livers of turkeys with hepatic lipidosis. Lipids Health Dis. 2017; 16: 98. PubMed Abstract \| Publisher Full Text\n\nSiddique A, Nelson J, Aouizerat B, et al.: Iron Deficiency in Patients with Nonalcoholic Fatty Liver Disease Is Associated with Obesity, Female Gender, and Low Serum Hepcidin. Clin. Gastroenterol. Hepatol. 2014; 12: 1170–1178. PubMed Abstract \| Publisher Full Text\n\nBritton LJ, Subramaniam VN, Crawford DH: Iron and non-alcoholic fatty liver disease. World J. Gastroenterol. 2016; 22: 8112–8122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDu F, Higginbotham DA, White BD: Food intake, energy balance and serum leptin concentrations in rats fed low-protein diets. J. Nutr. 2000; 130: 514–521. PubMed Abstract \| Publisher Full Text\n\nPezeshki A, Zapata R, Singh A, et al.: Low protein diets produce divergent effects on energy balance. Sci. Rep. 2016; 6: 25145. PubMed Abstract \| Publisher Full Text\n\nMiyatani S, Okuda T, Koishi H: Basal metabolism of Papua New Guinea highlanders. Japanese J. Phys. Fit. Sports Med. 1988; 37: 296–302.\n\nMaurer J, Taren DL, Teixeira PJ, et al.: The psychosocial and behavioral characteristics related to energy misreporting. Nutr. Rev. 2006; 64: 53–66. PubMed Abstract \| Publisher Full Text\n\nOlendzki BC, Ma Y, Hébert JR, et al.: Underreporting of energy intake and associated factors in a Latino population at risk of developing type 2 diabetes. J. Am. Diet. Assoc. 2008; 108: 1003–1008.\n\nSawaya AL, Tucker K, Tsay R, et al.: Evaluation of four methods for determining energy intake in young and older women: comparison with doubly labeled water measurements of total energy expenditure. Am. J. Clin. Nutr. 1996; 63: 491–499.\n\nMiyake M, Ogawa Y, Yoshida Y, et al.: Seven-year large cohort study for the association of serum albumin level and aging among community dwelling elderly. Journal of Analytical Bio-Science. 2011; 34: 281–286.\n\nGomi I, Fukushima H, Shiraki M, et al.: Relationship between serum albumin level and aging in community-dwelling self-supported elderly population. J. Nutr.Sci.Vitaminol. 2007; 53: 37–42. PubMed Abstract \| Publisher Full Text\n\nYamamoto M, Adachi H, Enomoto M, et al.: Lower albumin levels are associated with frailty measures, trace elements, and an inflammation marker in a cross-sectional study in Tanushimaru. Environ. Health Prev. Med. 2021; 26: 25. PubMed Abstract \| Publisher Full Text" }	[ { "id": "122201", "date": "09 Feb 2022", "name": "Dina Keumala Sari", "expertise": [ "Reviewer Expertise Nutrition", "vitamin and mineral" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 good research and finds the goals straight away and reaches the hypothesis of the study. This study also reports a new insight about how the adaptation mechanism can lower albumin levels.\nPlease describe how the sample (sampling method) was obtained, was it using purposive sampling or another sampling method?\nThere was no difference in albumin serum levels between the two groups, but please explain how long the adaptation mechanism lasted and how long it will last, based on this research or previous studies. Based on this analysis, we can also understand sago or rice's superiority.\nIn this study, the author did not separate between men and women, which can have different Hb (hemoglobin), suggestions to put 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": [] }, { "id": "209798", "date": "10 Oct 2023", "name": "Mihaela Ciulei", "expertise": [ "Reviewer Expertise My area of expertise is maternal and child nutrition with the focus on the micronutrient iron. More recently", "I have been examining the role of balanced energy-protein food/supplementation given along multiple micronutrients in pregnant or lactating women from low-resource settings on maternal and infant outcomes." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the cross-sectional study by Syauki et al. with great interest. I understand that this is the first study to analyze the protein-energy nutritional status of two adult populations of similar background who consumed sago with moderately low protein (PRO) intake vs rice with sufficient PRO intake. Below I separated my feedback in major and minor revisions. Overall, the Results and Discussion sections require the most improvements, and I tried to provide specific feedback on how these sections can be improved.\nMajor revisions\nBACKGROUND:\n“Mimika is classified, …higher risk of food insecurity” compared to what?\n\nThere are a lot of details with respect to the background of the food staple, sago. The introduction could benefit from more studies on the association between protein and food staples such as sago vs rice and health consequences if adults consume a moderate vs sufficient PRO diet.\nMETHODS:\nNo citation for the parent trial that focused on gut microbiota\n\nNo reasoning for the sample size calculation or a reference if this was detailed in the parent trial\n\nSection 2.1.4. no rationale indicated for why the complete blood count and plasma albumin were examined.\nRESULTS:\nSection 3.1.\nWhy is education background, occupation, income, etc. not part of Table 1?\n\nIt would be helpful to see the income also being indicated in US $.\n\nIt would be helpful to compare the information in Table 3 and 4 between the groups examined. For example, did the sago group consume significantly higher amounts of sweet potatoes? Also, it would be helpful to group the food sources based on groups recommended by prior literature like they did with green leafy vegetables. Then, compare statistically the groups of foods by the assigned sago vs rice groups.\n\nSection 3.2: Table 6: what is the prevalence of anemia in this population based on each anemia biomarker indicated in this table and what are the cutoffs used (this needs to be detailed in the METHODS as well)? Within text, authors indicate biomarker below or lower than the reference values, what are the reference values (these should be indicated in the Methods)? Why are only certain lab and not all values shown by men vs women?\nDISCUSSION:\nThe authors bring up studies that looked at consumption of sago, which are very helpful. It would be helpful to see some of these studies cited in the INTRODUCTION as well.\n\nOverall, this is a very lengthy discussion section, thus I highly recommend condensing the information, especially when the same information is included in multiple paragraphs.\n\nWhat does the minimum intakes for nitrogen equilibrium mean? This following sentence and paragraph is hard to follow. If authors want to compare the protein intakes with the recommendations, then they should indicate that from the first sentence of the paragraph. And then provide an explanation why and how their findings differ from the prior evidence, “The minimum intakes for nitrogen equilibrium were the main factors of the lower limits of successful adaptation at which an appropriate body composition can be maintained.”\n\n“Stunting was a nutritional problem in Indonesia.” Based on the national data referenced, it appears that stunting is still a problem in Indonesia. Is this an aspect that this manuscript tries to measure and address? I think not, as such, authors should not emphasize this point.\n\nSerum iron was not presented and measured in this study, thus authors should not make claims such as “In this study, lower iron levels were marked by a lower level of MCV.” The biomarkers assessed get at anemia status or type of anemia and not necessarily iron status.\n\n“Our study found that body composition and serum albumin levels were not significantly different between the groups of lowlanders of Mimika, Papua, Indonesia. Multivariate linear regression showed that there were different predictors of albumin between the sago-eating group and the rice-eating group. An adaptation mechanism for the sago-moderately low protein intake may have maintained the albumin levels within the normal range.” This is a nice and appropriate paragraph. What are the proposed adaptation mechanisms that the authors are speculating? Here it is a good place to indicate them.\n\nThe limitations of the study are clearly indicated, the strengths are not explicitly indicated.\nCONCLUSION:\n“Low levels of MCV (iron deficiency)” – low levels of MCV are not only indicative of iron deficiency anemia but can be a marker of other anemias as well (Reference book: Maner, Brittany S., and Leila Moosavi. \"Mean corpuscular volume.\" (2019). As such, authors should refrain from making iron deficiency claims. Again, iron metabolism is not the focus of the lab assessment but anemia is, as such authors should correct this observation throughout the paper. They can also speculate the cause of anemia to be iron deficiency, but without measuring specific iron biomarkers, there is no certainty.\nMinor revisions\nABSTRACT: Background- “Sago…is eaten with less protein.” Conclusion – “between”\nMETHODS: Statistical analysis section (and in the Results section), p-value not P-value\nRESULTS:\nSection 3.2\nSentence after Table 5: was instead of showed\n\nThe last sentence is missing a verb\n\nNeed a period before Serum albumin levels\n\nTable 6, It should be men and women (collective nouns) rather than man and woman (singular); footnote should indicate the statistical test used to compare categorical values and need to define all acronyms within table such as MCV.\n\nSection 3.3\nThe following information should be indicated in the Methods section for rationale and then can be brought up again in the Discussion but not in the results, “(the software was set to maintain variable at least P-value = 0.1) because hemoglobin and albumin were found to be correlated in anemic patients. While albumin and white blood cell were reported to be correlated in diabetic patients.”\n\nTables 7 & 8, what does B mean? The footnote of these tables should include the statistical test and all acronyms defined.\n\nThis sentence needs edited, “In the sago group, there were significant correlations between with the MCV and visceral fat (r = 0.415, P = 0.039) and between the visceral fat and basal metabolic rates (r = 507, P = 0.010).\n\nDISCUSSION:\nNeed a comma after rice cooked, “Based on the Indonesian Nutrient Composition Food, while sago per 100 g edible portion had a higher carbohydrate content than white rice cooked (85.6 g vs. 26.0 g), it had a lower protein content (0.6 g vs. 2.4 g).16”\n\n“Mimika district had better stature…” If authors aim to compare Mimika with the rest of the country, than the tense of the verb should be present tense, has.\n\nAuthors should not re-state the findings from the Results section, “Serum albumin levels in the sago group did not differ from those in the rice group and fell within the normal range. Multivariate linear regression analysis showed different predictive factors for albumin levels in these groups. Hemoglobin and white blood cells were factors related to albumin in the rice group, while MCV was a predictor of albumin in the sago group.“ Instead, they should indicate the main finding, what it means, and how it is similar/different from other findings in the relevant literature.\n\nThese sentences need references, “A moderately low protein intake does not change the basal metabolic rate or energy expenditure. Serotonergic and β -adrenergic systems are believed to be involved in the mechanism of the changes in energy balance. These are only two of the neurotransmitter systems involved in regulating food intake. Moderately low protein-high carbohydrate increases adiposity and fat in the liver. In an animal study, the lowest protein consumption increased the proportion of energy deposited as carcass fat. The higher the fat in the liver, the lower is the iron in the liver tissue. The higher the fat in the liver, the lower is the iron in the liver tissue. In this study, lower iron levels were marked by a lower level of MCV. This condition induces an adaptation mechanism of protein metabolism to maintain body protein balance (albumin within normal range).”\n\nFigure 1 is not necessary and authors may consider to remove it. All the speculated information is indicated within text but should have appropriate references.\n\nThe 2 paragraphs that start with “The rate of albumin synthesis…” and “Muscle mass…” seem to indicate evidence for the low protein intake in this study. Authors should guide the reader from the beginning of the paragraph by indicating that a potential explanation for these findings may be….\n\n“In our study, sago-eating participants with a moderately low protein diet showed more visceral fat (liver fat) than rice-eating participants.” Based on the results, there are no significant differences between visceral fat using body composition measures, as such authors should not make this claim.\n\nThis information is irrelevant as the current study is not assessing different methods of intake, “ A study by Sawaya, A.L. et, al in 1996 on comparing four different methods of the dietary survey such as a 7 day-weighing method, a 24-hour food recall (in duplicate), and two types of food intake frequency survey among young and older women with doubly labeled water (DLW) found that the total energy intake (TEI) by the 24-hour food recall method was the closest to the total energy consumption calculated by DLW for young women. The TEI/DLW was 86.7 percentage for young women (mean age 25.2 ± 3.5 years) and for older women (74.0 ± 4.4 years) was 75.2 percentages.48”\n\nThis information is not relevant in the current study, “This implicated on the production of short-chain fatty acid (SCFA) mainly butyrate as the preferred energy source for colonocytes.18” and “This implicated to have a higher production of short-chain fatty acid (SCFA) mainly butyrate, the energy source for colonocytes in the sago-eating people than rice-eating people.18 “ and “This study was a part of the main study on analysis of gut microbiota of rice-eating people with sufficient protein intake and sago-eating people with low protein intake of the lowlanders in Mimika Regency, Papua, Indonesia. We are interested in their habitual low protein intake of sago-eating people, but they appeared to be healthy. “\n\nBased on this sentence, “Another limitation in our study was the limited number of indicators for assessing the energy-protein nutritional status such as urinary excretion of nitrogen per unit of creatinine to measure nitrogen losses.” The reader may infer that urinary excretion of nitrogen was measured. Authors may want to indicate what was measured and what would have been better to measure and why, or what are the limitations of the current measures in the study, if that is the point they want to convey.\n\n“some participants in the rice group had better socioeconomic status,” was that a statistically significant difference between overall groups or just an observation?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-138
https://f1000research.com/articles/10-837/v1	20 Aug 21	{ "type": "Research Article", "title": "Effect of tuberculosis training on community health workers’ knowledge: a cluster randomized control trial in South Nigeria", "authors": [ "Christie Akwaowo", "Idongesit Umoh", "Oluseyi Motilewa", "Victor Umoh", "Eno Usoroh", "Stella Adeboye", "Uduak Idiong", "Etop Antia", "Idongesit Umoh", "Oluseyi Motilewa", "Victor Umoh", "Eno Usoroh", "Stella Adeboye", "Uduak Idiong", "Etop Antia" ], "abstract": "Background: Intensified efforts to improve tuberculosis (TB) diagnosis, treatment, and prevention are needed to meet global EndTB targets. Community health workers’ (CHWs) knowledge with respect to case finding is vital in tuberculosis elimination. This study aimed to determine the effect of tuberculosis training on the knowledge of community health workers in Nigeria. Methods: As part of a larger multicomponent intervention study, a randomised control trial was conducted with CHWs in 18 primary health care (PHC) clusters in Nigeria. The clusters were allocated to three arms: training and cash incentive (A), training only (B), and control (C) arms. Arms (A) and (B) received training on tuberculosis symptoms, prevention, diagnosis and treatment while the control arm (C) did not receive training. Participants’ knowledge on tuberculosis was assessed using questionnaires administered pre- and post-intervention. Data was analyzed using GraphPad Prism. Descriptive data was presented in tables and bivariate data was analyzed using chi square. Statistical significance was set as P<0.05. Results: There was a significant increase in the total knowledge score (25.4%), knowledge of general symptoms (24.4%), prevention (22.6%) and diagnosis and treatment (30.0%) across all study arms post-intervention (p<0.0001). Compared with the control arm, the training arm (A) had a higher proportion of good total knowledge score (94.4%) and arm (B) had a lower proportion of good total knowledge score (83.1%) when compared to the control group (93.1%). These were, however, not statistically significant. Conclusions: An improvement in the CHWs’ knowledge of symptoms, prevention, diagnosis and treatment of tuberculosis was observed after a training intervention was done. Integration of routine tuberculosis training is recommended to improve tuberculosis case finding in high burden communities. Pan African Clinical Trial Registry registration: PACTR202010691865364 (14/01/2020)", "keywords": [ "Tuberculosis", "Community health workers", "Patent medicine Vendors", "Training", "Knowledge" ], "content": "Introduction\n\nOne of the United Nations’ Sustainable Development Goals (SDG) as contained in the SDG target 3.3 is to end the tuberculosis epidemic by the year 2030. This End TB Strategy defines as a targets for 2030, a 90% reduction in the number of TB deaths and an 80% reduction in the TB incidence rate (new cases per 100 000 population per year) compared with levels in 2015.1 Progress in global TB elimination which has been inconsistent in recent years has led to intensified efforts to improve TB diagnosis, treatment, and prevention required to meet global targets for 2020–2035.2 To achieve these set targets, there’s a need for various national tuberculosis programs to have agendas set and implemented towards meeting goals. In 2019 alone, an estimated 1.4 million people died from TB-related illnesses and of the estimated 10 million cases that year, some 3 million went undiagnosed, or were not officially reported to national authorities.3\n\nDiagnosis, treatment and prevention of tuberculosis rests largely on the healthcare workers. Health care workers knowledge and attitude with respect to diagnosis, treatment and prevention of TB is vital in TB elimination.4 In Nigeria, the treatment of TB is free at all public health facilities. Successful implementation of directly observed treatment short course (DOTS) is dependent on the ability of the health care system to identify and properly manage TB cases which requires active involvement of the health workers in TB diagnosis and management. TB treatment should include counselling regarding progression of disease and the importance of treatment adherence.5\n\nCountries with good TB knowledge scores have been shown to have better TB indices with respect to treatment and prevention. In Iran, 98.4% of healthcare workers had a 'good' score for knowledge of TB with 98.2% who correctly answered that TB transmission is through the respiratory tract, 90% acknowledged TB is a treatable disease, and only 12.6% of them were oblivious that it is caused by a bacterium.6 In South India, accurate knowledge of TB was displayed by 86% of healthcare workers.7 Good and fair level of knowledge of TB was possessed by 56% and 43.9% of healthcare workers in Thailand.8 Studies in South Africa and Mozambique also reported satisfactory level of knowledge of TB among health care workers.4,9 In Mozambique, higher knowledge scores were seen among those with greater educational attainment. Knowledge scores were also affected by profession with medical doctors having the greatest knowledge score and midwives having the lowest knowledge scores.4\n\nA study in Vietnam also showed that an increased level of TB knowledge among the health staff is correlated with participation in TB training and higher medical education.10 In South Africa, the mean percentage of correct answers to the 98-item questionnaire administered was 59.5% pre-training and rose to 66.5% after training. Post-training, nurses, doctors and TB support staff showed significant improvements in total mean knowledge scores.11 A study in China that assessed knowledge retention of multidrug-resistant tuberculosis (MDR-TB) health practitioners 1 year after training, reported an overall positive long-term impact of the training on participants’ knowledge.12 Akande et al., in a study in Nigeria reported a significant increase in the knowledge of TB infection control after the health workers had been trained.13\n\nTraining of frontline health workers is an important strategy for TB control as efficient human resources development is crucial for facilitating tuberculosis control. In an ideal setting, training on TB should be integrated into the general education of health workers and into health systems. However, in a tuberculosis-endemic and resource-poor country like Nigeria, these systems are too weak to support routine effective tuberculosis control and training services.14 This study therefore aims to determine the effect of TB training on the knowledge of community health workers in Akwa Ibom state. This will aid in policy making and program designing with regard to TB control in the state.\n\n\nMethods\n\nThis study was registered as a clinical trial with the Pan African Clinical Trial Registry (PACTR202010691865364 on 14th October 2020). This article is reported in line with the Consolidated Standards of Reporting Trials (CONSORT) guidelines.27\n\nAkwa Ibom State is located in the Southern part of Nigeria, with a population of about 5.4 million people.15 It has 31 local governments across three senatorial districts. It has 368 primary health centers, unevenly distributed across the local government areas (LGAs). Akwa Ibom state has a high burden of TB and human immunodeficiency viruses (HIV). At the time of this study, the USAID was carrying out TB control activities in 15 LGAs and TB Reach had projects in 3 of the remaining 16 LGAs. The study population was the 13 LGAs that were not covered by the ongoing TB programs.\n\nThe study was designed as a three-arm parallel cluster randomized control study. We evaluated the effect of cash incentives and training on community health workers’ knowledge of TB. The PHCs were grouped in three clusters as shown in Figure 1.\n\nCHW = community health workers.\n\nA panel survey was planned but this was not possible as there was large in-and out migration of the patent medicine vendors (PMVs), especially those who originally sent their apprentices to the training. All the HCWs trained in the clusters were followed up for the duration of the study. At endline, we deployed the same method used in recruitment at baseline, targeting all HCWs in the study clusters. This design was used to minimize sampling error and take into account the design effect and prevent contamination across the three study arms. Therefore, participants were selected as two independent cross-sectional samples.\n\nThe interest in evaluating both training and cash incentives concurrently informed the choice of a multi-arm cluster randomized trial. It is also documented that sharing a control arm reduces the sample size relative to performing separate 2-arm trials.16 Cluster randomization was used in this study because the target of intervention was not individual health workers, but the PHC clusters. Each PHC cluster consisted of the PHC facility and its catchment communities. The RCT was conducted in six high TB burden LGAs in the state. The LGAs were selected from the sampling frame of 13 LGAs, with the aid of the State Tuberculosis and Leprosy Control Officer (STBLCO). In each participating LGA, 3 PHC facilities offering DOTS treatment and services were selected by simple random sampling and included in the study, each LGA has 3 clusters. Each PHC plus the catchment communities served as a cluster for this study. All community Health workers (CHWs) (PHC workers and patent medicine vendors) in each cluster were recruited into the study.\n\nThe selected LGAs were randomized to one of the three experimental arms by three researchers. Each researcher represented an intervention arm and picked from a box containing the names of the LGAs. Each LGA was assigned to the intervention arm picked by the researcher. All CHWs in the study clusters in each LGA were automatically assigned an intervention arm based on this randomization. Training with cash incentives was study cluster arm A and training only was arm B. The CHWs were recruited with the aid of the facility focal persons, working with the Chairman of the PMVs. All CHWs in the catchment were given an invitation to the training. The Field Coordinator conducted visits to the Facility Heads and Chair of PMV for each cluster. Formal letters were served to the Head of facility in each cluster. He was requested to mobilize all the CHWs in the catchment. He thus directed the Field Coordinator to the Chair of the PMVs who subsequently mobilized all the PMVs for the study. No exclusions were made, except if the person declined to participate. All CHWs in each cluster who participated in the workshop and gave consent were enrolled for the study under Arm A or B depending on their cluster.\n\nBlinding of participants to their allocated arms was not possible. To ensure that participants were blinded to the intervention, the LGAs chosen were spread apart and not contiguous to reduce contamination. Blinding of assessors to the different arms was also not possible.\n\nA one-day training session was carried out for each of the arms separately. The allocation ratio for the three clusters was 1:1:1 as shown in Figure 2.\n\nThe training was conducted by the researchers in collaboration with the State Tuberculosis and Leprosy Control Program office. Prior to the workshop, a Training of Trainers was conducted by the Lead Researcher, AKSTBLP State Coordinator and researchers. The Workshop facilitators were trained on the course contents. This was to develop a shared understanding of the aims of the workshop and familiarize them with the contents of the workshop materials. Training manuals were developed specifically for the research and distributed to participants. Facilitators used both the training manuals and power point presentations developed from the manuals. Facilitators used participatory learning methods to deliver the course contents. The contents of the training was based on the module developed for active TB case finding through house-to-house search for community based organizations (CBOs) and CHWs by the National Tuberculosis and Leprosy Control Program (NTBLCP).17 The sessions included training on basic symptoms, misconceptions diagnosis and treatment of TB, identification of presumptive TB cases, sputum collection and transportation and linking TB patients to care and treatment.\n\nParticipants were also taught how to collect sputum samples by the State Laboratory Focal Person for TB. Sputum cups were donated by the STBLCP program, the researchers procured transportation media (plastic bowls that could contain 4 sputa cups). At the end of the training, each participant was given a container for transporting sputum, 4 sputa cups, a presumptive case referral booklet and information, education and communication (IEC) materials to be used in educating clients.\n\nParticipants were instructed to carry out community education campaigns, identify presumptive TB cases in communities, collect sputum and make referrals to the PHC for treatment. The control Arm C had 41 CHWs. They were also requested to organize outreach health education campaigns in the communities. The participants in intervention arm A (85) received a cash incentive of two hundred naira (USD0.78) for every presumptive case referred for screening.\n\nThe interventions were carried out between April 2019 and March 2020. The trial ended as scheduled after one year.\n\nData collection was carried out for a period of 12 months starting in April 2019 and ended in March 2020. Data collection was done via quarterly supportive supervisory visits (SSV) made to the trained Community Health Extension Workers (CHEWs), PMVs and CPs. During the quarterly SSVs, data was collected from the CHWs and compared with the facility TB register. The number of presumptive cases referred, and number of outreaches conducted during the quarter were documented.\n\nA pre-intervention assessment of CHWs knowledge on TB was done using a pre-tested self-administered structured questionnaire.27 This was a standardized and validated questionnaire used by the national TB program for the 2017 TB Prevalence survey in Nigeria. The questionnaires were administered just before the training workshop for study arms A and B. However, they were administered at the six PHC facilities for the control arm as there was no training conducted for them.\n\nA post-intervention assessment was also done using the same tool after 12 months, in the PHC facilities in each of the clusters.\n\nKnowledge of TB was a secondary outcome variable for this study. The total knowledge score was 21. Good knowledge was taken as scores above 11, and poor scores below 11. Knowledge of TB was assessed at individual level and at cluster level. The focus of this paper was the secondary outcome. The secondary outcome measure of knowledge was assessed and analysed both at the individual participant level and cluster level.\n\nData was collected and entered into a Microsoft excel spreadsheet, version 2013, then collated and analyzed using STATA version 13 and GraphPad Prism version 8. R is an alternative open access software that could be used. The statistician was blinded to the study allocation until the data set was ready for final analysis. Descriptive statistics were carried out using frequencies and percentages and presented using tables. Chi square/Fischer’s exact test was used to compare the groups. And mean difference as effect size within each group. A p value of less than 0.05 was taken as statistically significant.\n\nEthical Approval was sought and obtained from the University of Uyo Institutional Health Ethics Research Board (UUTH/AD/S/93/VOLXXI/253). Approval was also obtained from the State Ministry of Health Ethics Review Board (MH/PRS/99/VOL.5/511). Written and verbal informed consent was obtained from the individual CHWs, the heads of each PHC cluster and the Chairman of each Local Government PMV association. Only CHWs who consented to participate were recruited into the study. All presumptive cases identified were referred for screening and positive cases were linked up to the State TB program for treatment with DOTS.\n\n\nResults\n\nA total of 240 CHWs were recruited at baseline and 153 trained, 85 in arm A and 73 in arm B, while 82 were recruited in the control arm26. At the end of the trial 72, 77 and 72 were analysed in arms A, B and C, respectively, to give a total of 221. Over the 12-month period of intervention, some CHWs retired, relocated and transferred out of the catchment area.\n\nMost of the CHWs in this study were female (62.5%), aged 30 years or less (47.9%), and had secondary level of education (64.6%). PMVs made up the majority of the health workers (78.8%), and over half (69.2%) worked at patent medicine shops. Most had worked at their current position for 1-4 years (32.1%). There was statistically significant difference in the duration at current position (P = 0.035) and type of facility of CHWs (P = 0.000) across the three arms. Also, a significantly higher proportion of CHWs in the control group (43.9%) had previously attended a TB workshop and had access to TB guidelines (31.7%) compared to their counterparts in other groups (Table 1).\n\n* Statistically significant.\n\n† Fisher’s exact.\n\nDf = degrees of freedom.\n\nTable 2 shows the sociodemographic characteristics across the three arms post-intervention. Most participants were aged 31-40 years (44.3%), female (53.4%), had secondary level of education (64.3%) and had worked for 1-4 years at their current position (38.0%). The majority were PMVs (77.4%), and worked at patent medicine stores (52.9%). There was a statistically significant difference in the duration of current job of the CHWs across the three arms (P 0.006), and in the type of health facility across the three arms (P 0.001).\n\n* Statistically significant.\n\nDf = degrees of freedom.\n\nThere was a significant increase in the total knowledge score of the respondents at end line (P 0.0001), with the respondents at baseline having a mean knowledge score of 11.8 (3.2) and at end line 16.8 (2.5). There was also statistically significant difference in the different categories of knowledge tested at baseline and at end line (Table 3).\n\n* Statistically significant.\n\nSD = standard deviation; CI = confidence interval.\n\nTable 4 compares the knowledge of tuberculosis across the three arms of the study. There was statistically significant relationship seen between respondents’ knowledge of diagnosis (P 0.0001), knowledge of prevention (P 0.014) and the total knowledge score (P 0.0480); and the different study arms.\n\n* Statistically significant.\n\nSD = standard deviation; Df = degrees of freedom.\n\nPost-intervention, a higher proportion of the respondents in the control group had good knowledge of diagnosis and treatment (75%) and prevention (93.1%) compared to the training and cash incentives group (55.6% and 76.4%, respectively). These were statistically significant at P 0.0224 and 0.0096 respectively (Table 5).\n\n* Statistically significant.\n\n† Fishers exact.\n\nDf = degrees of freedom.\n\nAt endline, a higher proportion of the respondents in the control group had good knowledge of diagnosis and treatment (75%) and prevention (93.1%) compared to the training only group (42.9% and 77.1%, respectively). These were statistically significant at p=0.0109 and 0.0001, respectively (Table 6).\n\n* Statistically significant.\n\n† Fishers exact.\n\nDf = degrees of freedom.\n\nAt baseline, CHWs who were in the training and CCT arm had a mean total knowledge score of 13.8 (2.4), and at end line, 14.5 (2.1), and this difference was statistically significant (P 0.0001). The difference in mean general knowledge score at pre- and post-intervention was also statistically significant (P 0.0430) (Table 7).\n\n* Statistically significant.\n\n† Fishers exact.\n\nSD = standard deviation; CI = confidence interval; Df = degrees of freedom.\n\nTable 8 shows the knowledge score of the health care workers that were in the training only arm at baseline and at endline. The mean general knowledge score, prevention score and total knowledge score showed a statistically significant increase at the end of the study compared to baseline (P 0.0001, 0.0353 and 0.0001, respectively.)\n\n* Statistically significant.\n\nSD = standard deviation; CI = confidence interval; Df = degrees of freedom.\n\nIn the control arm, the mean total knowledge score increased by 2.1 (95% CI:1.3-2.9) at endline, and this increase was statistically significant (P 0.0001). Significant increases were also noticed in the different categories of knowledge assessed for at endline compared to baseline (Table 9).\n\n* Statistically significant.\n\nSD = standard deviation; CI = confidence interval; Df = degrees of freedom.\n\n\nDiscussion\n\nThis randomized control trial aimed to assess the effect of tuberculosis training on community health workers knowledge on TB in Akwa Ibom state. Overall, there was improvement in the respondents’ knowledge post-intervention, compared to the baseline knowledge. The CHWs were comparable in their socio-demographic characteristics at baseline and at endline. It is worthy of note that a significant proportion of CHWs in the control arm had previously received training on TB, and had access to TB guidelines. This may be due to the attention the state has been receiving as a result of the high prevalence of HIV/AIDS, and consequently TB.\n\nThis study found a significant increase in CHW’s TB knowledge post intervention when compared to the baseline. The highest increase was noticed in knowledge of prevention, followed by knowledge of general symptoms. At baseline, the mean knowledge score of the CHWs was just above the average, increasing by a score of three at end line assessment. This corroborates with others studies that found an increase in knowledge of TB immediately after training and even long after training was done.13,18,19 Training was found in Abia state, Nigeria, to not only improve knowledge among Health workers, but to also improve all indicators of the Finding TB cases Actively, Separating safely, and Treating effectively (FAST) strategy which include the time to diagnosis (the time between when patient presents to the health facility to when a diagnosis of TB is made), time to treatment which the time between making a diagnosis and commencement of TB treatment, number of presumptive TB and Drug Resistant TB (DRTB) cases identified and number of TB and DRTB cases commenced on treatment.20\n\nComparing the three arms of the study post-intervention, it was observed that knowledge of diagnosis, prevention and the total knowledge score was significantly different across the three arms. Furthermore, the control arm was seen to have a higher mean knowledge score when compared to the arms that received training. This may have been an effect of previous training that the health workers in the control arm had received, as well as the fact that they reported having more access to TB guidelines. In a similar study among HIV infected people in Minna, training was shown to significantly improve the knowledge of the participants in the intervention group compared to the control group.21 In contrast, a similar study by Rakhshani et al. reported a significant increase in end line knowledge of malaria in the intervention group when compared to the control group.22 More participants from the training and cash incentives arm in the present study however had good knowledge scores when compared with those in the control arm, though this finding was not statistically significant.\n\nThis study compared the knowledge of CHWs in each arm at baseline and at endline. Among the participants in the training and cash incentives arm and in the training only arm, there was a significant increase in the mean total score between the baseline and post intervention. This is similar to results obtained in Turkey where training intervention increased the primary healthcare workers’ knowledge on immunization. Furthermore, the study reported a significant increase in the vaccination coverage rate in the community after the training intervention.23 Training intervention is therefore be effective in increasing knowledge and effecting behavior change. This is corroborated by other studies.21,22\n\nTraining community health workers on TB is essential to achieving TB control as they are the frontline health workers in the communities and are at a better position to identify TB cases. In a study in Southern Mozambique, health workers were seen to have poor knowledge of TB, and consequently low practice competency.24 Wu et al. found a decline in health workers knowledge one year post intervention, compared to the immediate post intervention period, though it was still higher than the pre-intervention knowledge.18 This therefore implies that training of health care workers should not be a one-time event, but a continuous process if TB control is to be achieved. Experiences in Kyrgyzstan14 and the Democratic Republic of Congo show that periodic training and supervision in the field can improve healthcare workers’ TB knowledge and skills.25\n\n\nLimitations\n\nThis study had some limitations. Firstly, though this study was planned as a panel survey, this could not be carried out. This is because some of health workers in the different communities may have resigned or moved away, while others took up these positions. Therefore, not all those that were trained at baseline could participate in the post intervention assessment. Also, a few CHWs were transferred in and were part of the post intervention assessment since it was a cluster sampling.\n\nAnother limitation was the selection bias seen in the study. The control arm had more exposure to TB training as evidenced by the baseline characteristics like exposure to workshops and access to TB guidelines were significantly higher among the control group. The control arm was likely to have had previous trainings on TB and had access to TB guidelines which may have affected the study results. This is because random allocation of PHCs to the different arms was carried out prior to pre-intervention assessment.\n\n\nConclusion\n\nThis study was conducted to determine the effect of TB training on the knowledge of community health workers in Akwa Ibom state, Nigeria. Patent Medicine Vendors formed two thirds of the CHWs mobilized, indicating they were readily available in the communities. We found a significant improvement in the CHWs knowledge of symptoms, prevention, diagnosis and treatment of TB after the training intervention was done. We thus recommend the integration and use of PMVs in the delivery of interventions in the community. Furthermore, integration of routine TB training for all categories of CHWs will improve TB case finding and notification, improving TB indices in the communities and the country.\n\n\nData availability\n\nDryad: Improving Tuberculosis Case Finding in Akwa Ibom State, Nigeria. https://doi.org/10.5061/dryad.tht76hf07.26\n\nThe project contains the following underlying data:\n\n- Readme_Improving Tuberculosis case finding in Nigeria\n\n- TB community data Post intervention\n\n- TB Community data pre-intervention\n\n- TB Health care workers Baseline\n\n- TB Health care workers endline\n\n- TB study Outcome (summary)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication)\n\nZenodo: Improving Tuberculosis case finding in Nigeria. https://doi.org/10.5281/zenodo.5062448.27\n\nThis project contains the following extended data:\n\n- Ethical_Approval_2_SMOH.jpg\n\n- Ethical_Approval_TB_1.pdf\n\n- FINAL_RESEARCH_PROTOCOL._FOR_TB_STUDYdocx.docx\n\n- Questionnaires_for_the_TB_Study.zip\n\n- Training_Kit.zip\n\n- TrialApprovalLetter3.pdf\n\nZenodo: CONSORT checklist for ‘Effect of tuberculosis training on community health workers’ knowledge: a cluster randomized control trial in South Nigeria’. https://doi.org/10.5281/zenodo.5062448.27\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe Team acknowledges Mr. Edidiong Umoh and Mrs Ekom Ekwo of the Health Systems Research Hub, University of Uyo for their support in executing the entire project.\n\n\nReferences\n\nTuberculosis (TB). [cited 2021 Feb 3]. Reference Source\n\nMacNeil A: Global Epidemiology of Tuberculosis and Progress Toward Achieving Global Targets — 2017. MMWR Morb Mortal Wkly Rep. 2019 [cited 2021 Feb 3]; 68. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nGlobal Tuberculosis Report 2020 - World. ReliefWeb. [cited 2021 Feb 3]. Reference Source\n\nNoé A, Ribeiro RM, Anselmo R, et al.: Knowledge, attitudes and practices regarding tuberculosis care among health workers in Southern Mozambique. BMC Pulm Med. 2017 Dec;17(1):1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrganization WH: Global tuberculosis control: WHO report 2011. World Health Organization ; 2011 [cited 2021 Feb 3]. Reference Source\n\nHashim DS, Al Kubaisy W, Al Dulayme A: Knowledge, attitudes and practices survey among health care workers and tuberculosis patients in Iraq. EMHJ - East Mediterr Health J. 2003 [cited 2021 Feb 3]; 94: 718–731Reference Source\n\n(PDF) A systematic review of the epidemiology of and programmatic response to TB in people living in urban informal settlements in South Africa.[cited 2021 Feb 3]. Reference Source\n\nTemesgen C, Demissie M: Knowledge and practice of tuberculosis infection control among health professionals in Northwest Ethiopia; 2011. BMC Health Serv Res. 2014 Nov 19; 14(1): 593. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhebhe LT, Van Rooyen C, Steinberg WJ: Attitudes, knowledge and practices of healthcare workers regarding occupational exposure of pulmonary tuberculosis. Afr J Prim Health Care Amp Fam Med. 2014 Jan; 6(1): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoa NP, Diwan VK, Thorson AE-K: Diagnosis and treatment of pulmonary tuberculosis at basic health care facilities in rural Vietnam: a survey of knowledge and reported practices among health staff. Health Policy Amst Neth. 2005 Apr; 72(1): 1–8. PubMed Abstract \| Publisher Full Text\n\nNaidoo S, Taylo M, Esterhuizen TM, et al.: Changes in Healthcare Workers’ Knowledge about Tuberculosis Following a Tuberculosis Training Programme. Educ Health. 2011 Aug 1; 24(2): 514. PubMed Abstract\n\nWu S, Li R, Su W, et al.: Is knowledge retained by healthcare providers after training? A pragmatic evaluation of drug-resistant tuberculosis management in China. BMJ Open. 2019 Mar 1 [cited 2021 Mar 1]; 9(3): e024196. Publisher Full Text Reference Source\n\nAkande PA: The effect of an educational intervention to improve tuberculosis infection control among nurses in Ibadan, south-west Nigeria: a quasi-experimental study. BMC Nurs. 2020 Aug 28 [cited 2021 Mar 1]; 19(1): 81. Publisher Full Text\n\nAwofeso N, Schelokova I, Dalhatu A: Training of front-line health workers for tuberculosis control: lessons from Nigeria and Kyrgyzstan. Hum Resour Health. 2008 Sep 29; 6: 20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkwa Ibom (State, Nigeria) - Population Statistics, Charts, Map and Location. 2017 [cited 2020 Jan 6]. Reference Source\n\nDelea MG, Snyder JS, Belew M, et al.: Design of a parallel cluster-randomized trial assessing the impact of a demand-side sanitation and hygiene intervention on sustained behavior change and mental well-being in rural and peri-urban Amhara, Ethiopia: Andilaye study protocol. BMC Public Health. 2019 Jun 21 [cited 2020 Dec 11]; 19(1): 801. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFMOH: Participants’ Module For Community Based Organizations and Community Health Workers For Active TB Case Finding Through House-To-House Search. FMOH; 2015.\n\nWu C, Kao S-C, Shih C-H, et al.: Open data mining for Taiwan’s dengue epidemic. Acta Trop. 2018; 183: 1–7. PubMed Abstract \| Publisher Full Text\n\nNaidoo S, Taylor M, Esterhuizen T, et al.: Changes in healthcare workers’ knowledge about tuberculosis following a tuberculosis training programme. Educ Health Abingdon Engl. 2011 Aug 1; 24: 514. PubMed Abstract\n\nIwuoha EC, Onwasigwe CN: Improving “Fast” Indicators of TB Infection Control through Targeted Health Workers Training; Findings from Facility Based Studies in Abia State, Nigeria. Int J Trop Dis Health. 2020 Dec 26 [cited 2021 Mar 1]; 1–9. Reference Source\n\nBisallah CI, Rampal L, Lye M-S, et al.: Effectiveness of health education intervention in improving knowledge, attitude, and practices regarding Tuberculosis among HIV patients in General Hospital Minna, Nigeria – A randomized control trial. PLoS One. 2018 Feb 22 [cited 2020 Sep 14]; 13(2): e0192276. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nRakhshani F, Mohammadi M: Improving community health workers’ knowledge and behaviour about proper content in malaria education. JPMA J Pak Med Assoc. 2009 Jun; 59(6): 395–398. PubMed Abstract\n\nUskun E, Uskun SB, Uysalgenc M, et al.: Effectiveness of a training intervention on immunization to increase knowledge of primary healthcare workers and vaccination coverage rates. Public Health. 2008 Sep [cited 2021 Mar 1]; 122(9): 949–958. PubMed Abstract \| Publisher Full Text Reference Source\n\nNoé A, Ribeiro RM, Anselmo R, et al.: Knowledge, attitudes and practices regarding tuberculosis care among health workers in Southern Mozambique. BMC Pulm Med. 2017 Jan 5; 17(1): 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDriessche KV, Sabue M, Dufour W, et al.: Training health care workers to promote HIV services for patients with tuberculosis in the Democratic Republic of Congo. Hum Resour Health. 2009 Mar 17 [cited 2021 Mar 1]; 7(1): 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkwaowo C, et al.: Improving Tuberculosis case finding in Nigeria, Dataset, Dryad. 2021. Publisher Full Text\n\nAkwaowo, Christie, Umoh, et al.: Improving Tuberculosis case finding in Nigeria. Zenodo. 2021. Publisher Full Text" }	[ { "id": "93496", "date": "04 Oct 2021", "name": "Angela Oyo-Ita", "expertise": [ "Reviewer Expertise Public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis was a randomized control trial to study the effect of training and or incentive on the knowledge of community health workers. The participants were health workers in formal (community health workers) and informal (patent medicine vendors, PMVs) settings. The title could be amended to read “Effects of training of frontline health workers’ on tuberculosis: a cluster-randomized control trial in South Nigeria”.\nA succinct description of the intervention should be provided.\nWhat were the qualifications of the trainees? This is particularly needed as apprentices were sent by some of the PMVs for the training.\n\nHow many training sessions were held?\n\nWere the follow-up training sessions repeats of the initial training?\n\nWhat was the level of participation of the trainees?\n\nWhat training method(s) was adopted?\n\nThe average period of exposure of the trainees should also be indicated.\n\nThe attrition rate should be indicated and the reasons highlighted.\nThough the intervention sites were said to be contiguous, the result seems to indicate that there may be some level of contamination as the impact on the control was substantial.\nThe second socio-demographic table is not needful. Intention to treat analysis should be presented.\nDiscussion should proffer possible reasons for the increase in the knowledge in the control arm.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": [ { "c_id": "7315", "date": "02 Feb 2022", "name": "Christie Akwaowo", "role": "Author Response", "response": "This was a randomized control trial to study the effect of training and or incentive on the knowledge of community health workers. The participants were health workers informal (community health workers) and informal (patent medicine vendors, PMVs) settings. The title could be amended to read “Effects of training of frontline health workers’ on tuberculosis: a cluster-randomized control trial in South Nigeria”. Response: The title has been amended accordingly. What were the qualifications of the trainees? This is particularly needed as apprentices were sent by some of the PMVs for the training. Response: The trainees were Community Health Workers and patent medicine vendors. How many training sessions were held? Were the follow-up training sessions repeats of the initial training? Response: One training session was held. This has been highlighted in the paper. What was the level of participation of the trainees? What training method(s) was adopted? Response: A participatory method of training was adopted and the trainees were involved in the interactive session. This has been highlighted in the paper. The average period of exposure of the trainees should also be indicated. Response: This has been indicated in the paper. Though the intervention sites were said to be contiguous, the result seems to indicate that there may be some level of contamination as the impact on the control was substantial. Response: The intervention sites were non-contiguous, however, the possible source of contamination has been highlighted. The second socio-demographic table is not needful. Intention to treat analysis should be presented. Response: We beg to differ, we think that information captured on this table has not been replicated elsewhere and should be retained. Discussion should proffer possible reasons for the increase in the knowledge in the control arm. Response: This had been highlighted and also included in the limitations." } ] } ]	1	https://f1000research.com/articles/10-837
https://f1000research.com/articles/11-132/v1	02 Feb 22	{ "type": "Research Article", "title": "The role of oxidative stress markers in Indonesian chronic kidney disease patients: a cross sectional study", "authors": [ "Mochamad Yusuf Alsagaff", "Mochammad Thaha", "Budi Susetyo Pikir", "Hendri Susilo", "Citrawati Dyah Kencono Wungu", "Satriyo Dwi Suryantoro", "Mutiara Rizky Haryati", "Ristra Ramadhani", "Esthiningrum Dewi Agustin", "Muhammad Rifqi Arya Putra", "Masayuki Maiguma", "Yusuke Suzuki", "Mochamad Yusuf Alsagaff", "Budi Susetyo Pikir", "Hendri Susilo", "Citrawati Dyah Kencono Wungu", "Satriyo Dwi Suryantoro", "Mutiara Rizky Haryati", "Ristra Ramadhani", "Esthiningrum Dewi Agustin", "Muhammad Rifqi Arya Putra", "Masayuki Maiguma", "Yusuke Suzuki" ], "abstract": "Background: Several aspects of chronic kidney disease (CKD) such as the incidence rate and mortality rate are concerning. Oxidative stress contributes to progression and mortality in patients with CKD; however, a specific correlation between several markers of oxidative stress and the estimated glomerular filtration rate (eGFR) and albumin-creatinine ratio (ACR) in the Indonesian population has not been sufficiently described yet. Methods: This study was an analytic observational study with a sample of 56 patients with CKD in Universitas Airlangga Hospital, Surabaya, Indonesia, from December 2019 – March 2020. The markers for oxidative stress investigated were urinary 8-hydroxy-2 deoxyguanosine (8-OHdG), serum symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA). The correlations between each variable of oxidative stress and CKD were analyzed using Pearson analysis. Results: There was a positive correlation between 8-OHdG and eGFR (p=0.00, r=0.51); however, there was a negative correlation between 8-OHdG and ACR (p=0.025, r=-0.30). SDMA and eGFR showed a negative correlation (p=0.00, r=-0.648), while SDMA and ACR showed a positive correlation (p=0.03, r=0.349). ADMA showed a negative correlation with eGFR (p=0.00, r=-0.476). There were significantly decreased 8-OHdG but increased ADMA and SDMA as the CKD stage progressed (p=0.001, p=0.00, and p = 0.00, respectively). Higher urine 8-OHdG was detected in patients without history of hemodialysis, whereas ADMA and SDMA showed higher value in patients with hemodialysis (p=0.00, p=0.00, and p=0.004, respectively), patients with history of diabetes mellitus (DM) had higher mean 8-OHdG (p 0.000) yet lower serum ADMA and SDMA (p=0.004 and p=0.003, respectively). Conclusions: In patients with CKD in Indonesia, the markers for oxidative stress 8-OHdG, SDMA, and ADMA are correlated with eGFR and ACR levels. There were also significant difference in 8-OHdG, SDMA, and ADMA levels among CKD stages, between dialysis vs non dialysis, and DM vs non DM patients.", "keywords": [ "oxidative stress", "chronic kidney disease", "8-OHG", "SDMA", "ADMA", "health risk" ], "content": "Introduction\n\nChronic kidney disease (CKD) is one of the diseases with the highest incidence and mortality rates among noncommunicable diseases. There were 697.5 million cases of all-stage CKD, and 1–2 million people died from CKD in 2017.1 A study in Korea found that the mortality rate of patients with CKD was 134 per 1000 patients annually and higher than that for patients with diabetes mellitus (DM) or hypertension without CKD, which was only 34 per 1000 patients annually.2 This result showed that CKD is a health burden with a high cost for the health care system in both developed and developing countries, including Indonesia. According to the results of Basic Health Research in 2018, 0.38% or 739,208 Indonesian citizens suffer from CKD.3 This number is increased compared to the result of Basic Health Research in 2017, which found that the prevalence of CKD at the time was 0.2%.4\n\nOxidative stress contributes greatly to the progression and mortality of patients with CKD. In a previous study, 8-Hydroxy 2-deoxyguanosine (8-OHdG) was found to have correlations with an increased risk of death in patients with CKD with different levels of the estimated glomerular filtration rate (eGFR).5 Asymmetric dimethylarginine (ADMA), which inhibits nitric oxide both in vivo and in vitro, is a strong predictor of progression in patients with CKD stage 3–4 (eGFR between 25–40 mL/min/1.73 m2).6 An increased level of symmetric dimethylarginine (SDMA), which also has a similar function as ADMA, is closely correlated with an increased risk of death in patients with CKD.7 Several studies have shown that there is a correlation between the levels of oxidative stress markers and CKD in general; however, studies in the Indonesian population that showed the relationship between these variables are still very limited. Therefore, we decided to study the correlation between 8-OHdG, SDMA, and ADMA with the level of severity of CKD as measured by albumin-creatinine ratio (ACR) and eGFR.\n\n\nMethods\n\nThis study was an analytic observational study with a cross-sectional design. This study used the consecutive sampling technique. The sample contained 56 patients with CKD in the Nephrology Outpatient Clinic, Universitas Airlangga Hospital, Surabaya, Indonesia who met the inclusion criteria from December 2019 to March 2020.60 The inclusion criteria of this study selected patients with CKD of all stages who were under medical care in Universitas Airlangga Hospital aged 21 years and older. The exclusion criteria of the samples in the study removed patients with a history of acute coronary syndrome, acute heart failure, severe infection, cancer, and arrhythmia.\n\nWritten informed consent to be a study subject was obtained from the patients according to the Declaration of Helsinki. This study received approval from the ethical committee of Universitas Airlangga Hospital (certification number: 189/KEH/2019).\n\nData was obtained through history taking, anthropometric and vital sign examinations, and blood and urine sampling from the patients. History taking was carried out by directly interviewing the patients: Basic identity, duration of kidney disease, duration of dialysis, history of hypertension, history of diabetes mellitus, history of smoking and history of cardiovascular disease were all taken into consideration. Anthropometric examination was carried out to measure height, body weight, and abdominal circumference. Vital sign examination of blood pressure was carried out using a mercury sphygmomanometer (Big Ben stand model, Riester, Germany). eGFR measurement was carried out using the CKD-EPI Creatinine Equation (2009) to estimate the glomerular filtration rate (GFR).8 A 6-cc blood sample was drawn from the patients, the serum was separated, and urine samples were also obtained for laboratory marker examination in a private laboratory.\n\nMeasurement of 8-OHdG, ADMA, and SDMA levels was carried out with the enzyme-linked immunoassay (ELISA) method using a Human 8-OHdG ELISA Kit (Cat. No E-EL-0028, Elabscience, USA), Human ADMA ELISA Kit (Cat. No E-EL-0042, Elabscience, USA), and Human SDMA ELISA Kit (Cat. No E-EL-H5659, Civic Bioscience, Canada), respectively. HbA1c levels were measured using high-performance liquid chromatography using an HbA1c HPLC assay (Cat. No A1C31-H100, Eagle Bioscience, USA). Lipid profile [total, low-density lipoproteins (LDL), and high-density lipoproteins (HDL) cholesterol], albumin, and serum creatinine were performed in fasting patients and conducted in Prodia laboratory, Surabaya, Indonesia.\n\nUrine samples were taken from patients with all CKD stages, including patients who had undergone chronic dialysis (5D) after dialysis. Each patient was given a urine sample tube to take home in order to collect the urine. The urine samples were checked for 8-OHdG by ELISA using the Human 8-OHdG ELISA Kit (Cat. No E-EL-0028, Elabscience, USA), the albumin was measured with a calorimetry method9 using an Albumin (BCG) Assay Kit (Colorimetric) (Cat. No ab235628, Abcam, UK) through the utilization of bromocresol purple that forms a colored complex specifically according to the manufacturer’s instruction, and urine creatinine was measured with a colorimetry method using a creatinine assay kit (Cat. No MAK080, Sigma Aldrich, USA). From the albumin and urine creatinine levels, we calculated the albumin-creatinine ratio (ACR).\n\nNumerical data are presented as the mean ± SD. Data normality tests were carried out using the Shapiro–Wilk test. Pearson correlation tests were used to analyze the correlation between each variable. Kruskal Wallis followed by Mann Whitney post hoc test was conducted to determine the differences of 8-OHdG level in each CKD stage. One-way ANOVA followed by post-hoc LSD test was performed to determine the differences of SDMA and ADMA levels in each CKD stage. The Mann Whitney test was used to analyze the differences in 8-OHdG and SDMA levels between dialysis vs non dialysis patients and DM vs non-DM patients. Independent t-test was performed to determine the association in ADMA levels between dialysis vs non dialysis patients and DM vs non-DM patients. A p value of <0.05 showed a significant test result. All statistical analysis was performed using SPSS application version 23 (IBM Corporation, Armonk, New York, USA).\n\n\nResults\n\nThe characteristics of the study samples are presented in Table 1. The age range of the patients enrolled was between 31–71 years old, with a majority of subjects being male (55.4%) compared to female (44.6%). The sample was dominated by patients with CKD stage 5 (42.9%), in which 3.6% were predialysis and 39.3% were on dialysis. The majority of the study subjects had comorbid hypertension (89.3%). The majority of the patients had diabetic kidney disease (75%), while the cause of the other cases (25%) was not known. Anthropometric examination showed that the majority of the patients were categorized as obese (57%). Lipid profile tests also showed dyslipidemia in the majority of the samples with an increase in total cholesterol level and LDL cholesterol level, while a decrease in HDL cholesterol level. The oxidative stress marker examination found varied results.\n\na Percentage of total samples.\n\nb Percentage of total samples in each stage.\n\nAs shown in Table 2, we found a significant positive correlation between 8-OHdG and eGFRR (p=0.00, r=0.51); however, there was a negative correlation between 8-OHdG and ACR (p=0.025, 5=-0.30). SDMA and eGFR showed a negative correlation (p=0.00, r=-0.648), while SDMA and ACR showed a positive correlation (p=0.03, r=0.349). Meanwhile, ADMA showed a negative correlation with eGFR (p=0.00, r=-0.476), whereas ADMA and ACR were not significantly correlated. From Table 3, it can be inferred that there were significant differences of 8-OHdG, ADMA, and SDMA levels among CKD stages. As can be seen, urinary 8-OHdG significantly declined as the CKD progressed (p=0.001), with the lowest level in stage V. On the contrary, both ADMA and SDMA showed significant increase as the CKD got more advanced (p=0.000 and p=0.000 respectively), with the highest levels in stage V.\n\n* Statistically significant (p<0.05).\n\na Kruskal Wallis followed with Mann Whitney.\n\nb ANOVA followed with LSD.\n\n8-OHdG, ADMA, and SDMA were also evaluated among patients with previous history of hemodialysis and diabetes mellitus (Table 4). All of the stress oxidative markers tested above showed different results when being compared between patients with a history of hemodialysis and patients without (8-OHdG p=0.000; ADMA p=0.000; SDMA=0.000). Higher urinary 8-OHdG was detected in patients without history of hemodialysis, whereas ADMA and SDMA showed higher value in patients with hemodialysis. Patients with history of diabetes mellitus also had higher mean 8-OHdG (p=0.000) yet lower serum ADMA and SDMA (p=0.004 and p=0.003, respectively).\n\nx Mann Whitney.\n\ny Independent t-test.\n\n\nDiscussion\n\nGender and age\n\nAccording to the results of the study, the number of male patients (31/56, 55%) was slightly greater than that of female patients (25/56, 45%); however, there was no significant difference between gender and the prevalence of CKD. We also found that the average age of the patients was 57.02±7.59, which is in accordance with the increasing prevalence of CKD with age. Increasing age will affect the anatomy, physiology and cytology of the kidneys. The changes that occur, including a reduced number of nephrons due to chronic glomerulonecrosis, cause disturbances of physiological processes of the kidneys, characterized by a decrease in GFR as age increases.10\n\nCKD stage and history of hemodialysis\n\nThis study involved patients from all CKD stages.1–5 According to this study, the majority of patients were in stage 5 (42.8%), followed with stage 3 and 4 (23.2% for both), stage 2 (7.1%), and the least were stage 1 (3.6%). The number of patients with a history of hemodialysis was 22 (39%). Hemodialysis is an artificial process as a substitute of the kidney, removing wastes and excess water from the blood, mainly in patients with impaired renal function.11 Most guidelines recommend dialysis in CKD patients with a GFR < 15 ml/min/1.73 m2, while the role of hemodialysis in CKD patients with GFR above 15 ml/min/1.73 m2 is uncertain.12\n\nHistory of hypertension and diabetes mellitus\n\nThe majority of the patients had a history of hypertension (89.3%), with an average systolic blood pressure of 138.93±22.31 mmHg and diastolic blood pressure of 78.32±12.61 mmHg. From these results, we can see that the great majority of CKD patients suffer from hypertension, which is both a cause and an effect of the disease.13 Moreover, we also found a large number of patients had a history of diabetes mellitus (75%) with an average HbA1c level of 6.85±1.67%. This is in accordance with a study by Sinusi and Hargono (2021) according to data from the Indonesian Family Life Survey 5 (IFLS-5), which showed that hypertension and diabetes mellitus are the primary risk factors for CKD.14 Uncontrolled hypertension is a risk factor of arteriolar nephrosclerosis, a vascular injury in the preglomerular arterioles that leads to glomerular ischemia,15 whereas a high level of blood glucose will impair the insulin signaling pathway in the glomerulus of the kidneys, which leads to proapoptotic environment.16 Diabetic kidney disease (DKD) is the most common cause of the end-stage renal disease (ESRD). The prevalence of DKD remains high despite rigorous treatments such as hyperglycemic management, blood pressure control, and the use of renin-angiotensin system blockades.17 In DKD, increased proximal tubular reabsorption of glucose via sodium–glucose cotransporter 2 reduces distal supply of solutes, notably sodium chloride, to the macula densa. The consequent decrease in tubuloglomerular feedback may widen the afferent arteriole, increasing glomerular perfusion, whereas increased local angiotensin II production causes vasoconstriction at the efferent arteriole. These will lead to high intraglomerular pressure and glomerular hyperfiltration.18,19\n\nObesity and lipid profile\n\nFrom anthropometric examination, it was found that the majority of patients were categorized as obese (57%), with an average body mass index (BMI) of 26.08±4.65 and average abdominal circumference of 91.84±17.79 cm. This corresponds to a previous study that showed that there is an association between obesity and high blood pressure.20–22 The results of the lipid profile test of the patients also showed dyslipidemia in the majority of samples with increased total cholesterol levels (216.55±66.02 mg/dL) and LDL cholesterol (126.39±52.73 mg/dL) and decreased HDL cholesterol levels (44.41±15.70 mg/dL). Dyslipidemia is a common finding in patients with kidney disease, in which the abnormal lipid profile varies, such as high triglycerides and total cholesterol, increased LDL followed by low, normal, or increased HDL.23\n\n8-OHdG and chronic kidney disease\n\nOne of the risk factors for kidney damage is oxidative stress.24 One of the markers of oxidative stress found to be increased in patients with CKD is a product of nucleic acid oxidation, which is 8-OHdG.25 8-OHdG or 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) are free radicals commonly found due to oxidative injury and often become markers of oxidative stress or carcinogenesis.26 This biomarker is also helpful in detecting the presence of microvascular and macrovascular complications.27 8-OHdG is excreted in the plasma and urine; therefore, can be easily measured.28\n\nIn patients with CKD, there is an increased level of oxidative stress biomarkers, including 8-OHdG.29 In this study, there was a significant positive correlation between 8-OHdG and eGFR (p=0.00, r=0.51; moderate correlation); however, there was a negative correlation between 8-OHdG and ACR (p=0.025, r=-0.030; very weak correlation). This is in accordance with a previous study that also showed that increased levels of 8-OHdG were directly proportional to increased eGFR and inversely correlated with ACR.30 Another study by Dincer et al (2008) also showed an increased level of this biomarker in patients with proteinuria.31\n\nIn this study, there was a significant decline in 8-OHdG level among CKD stages (p=0.001). This is different from previous studies, which showed an increased level of oxidative stress biomarker, including 8-OHdG along with CKD progression.29,32 In contrast with SDMA and ADMA, which were measured from serum, 8-OHdG was measured from urine. A possible explanation was because renal function has a direct effect on the ability to filter and eliminate solutes like oxidant stress biomarkers. As a result, those with poor kidney function may have been the least able to filter biomarkers at the glomerular level and/or produce oxidative stress analytes into the urine. Low urine concentrations would arise from these abnormalities. In epidemiologic literature, this is referred to as reverse causation, because kidney function may alter the exposure measurements of interest.30 We also found a significantly lower 8-OHdG level in dialysis vs non dialysis patients (p=0.000). This is in line with a study by Navarro et al (2019), which stated that the membrane and dialysate cause inflammation and a significant rise in ROS generation when dialysis is first started. The levels of Oxidized LDL have been found to be higher after dialysis. However, post-dialysis, xanthine oxidase activity and 8-OHdG levels are significantly lower, implying that oxidative stress indicators are efficiently filtered during the dialysis process, suggesting that dialysis could reduce oxidative stress markers33 We also found a significantly higher 8-OHdG level in DM vs non-DM patients (p=0.000). A study conducted by Liu et al (2016) found significantly increased 8-OHdG in patients with type two DM with and without complications compared to healthy control. Increased levels of 8-OHdG as an oxidative stress marker are likely to cause strand breakage and oxidative base alterations, and numerous signaling pathways may potentially play a role in glucotoxicity's negative effects on cellular activities.34\n\nADMA and chronic kidney disease\n\nADMA is a potent endogenous nitric oxide synthase (NOS) inhibitor. This substance can accumulate and cause endothelial disorders, increased blood pressure, and proteinuria which contribute to the progression of cardiovascular diseases leading to kidney dysfunction.35 Over synthesis of ADMA causes increased blood pressure, extracellular matrix synthesis, and decimation of peritubular capillaries, which may result in chronic kidney failure.36 Previous studies showed that there was an increase in ADMA levels due to inhibition of nitric oxide (NO) discharge in patients with CKD accompanied by decreased endothelial function. ADMA competes with L-arginine to inhibit NOS, which causes endothelial dysfunction.37 Several functions of NO in the body are the regulation of vascular tone and blood pressure; therefore, if there is an inhibition of NOS, the regulation of both of these systems will also be compromised.38 A previous study showed proof of the impact of increased ADMA levels on the cardiovascular risk of patients with CKD, which was a significant positive correlation between increased ADMA levels and carotid intima-media thickness; therefore, this biomarker can also detect atherosclerosis in patients with CKD.39\n\nThis study showed that there was a negative correlation between ADMA and eGFR (p=0.00, r=-0.476; moderate correlation). This is in accordance with other studies that also found that increased plasma ADMA concentration has a significant negative correlation with a decrease in eGFR; however, the exact mechanism is still unclear.40 In this study, we found that significantly increased ADMA level was found in late CKD stage and in dialysis compared to non-dialysis patients (p=0.000 for both). As stated by previous studies, increased levels of ADMA are associated with the progression of CKD, especially in those who received routine hemodialysis.41–43 Vallance et al. also found that hemodialysis patients with ESRD had higher ADMA levels than controls. ADMA appears to predict cardiovascular outcome and mortality in ESRD dialysis patients.44 However, there are other independent factors that should be taken into consideration, including age, sex, and smoking history.45 Another factor that should not be overlooked is genetic factors. Previous studies have shown that increased ADMA levels are also associated with certain genetic variations, primarily the G-449 allele in the DDAH2 gene.46 We also found a significantly increased ADMA level in DM patients compared to non DM (p=0.004). In type 2 DM, ADMA may play a key role in increasing vascular damage and is a strong determinant of insulin resistance.47\n\nThis study also showed that there was no significant correlation between ADMA and ACR (p=0.278). This result contradicts previous studies that found that there is an association between ADMA level and ACR. Several mechanisms by which this association occurs are inflammatory processes, vascular disorders such as endothelial dysfunction or atherosclerosis, and other processes, namely, collagen production and glycation processes in the renal microcirculation.48,49 However, these associations cannot be separated from other factors, such as distinct individual demographic characteristics, hemodynamics and metabolic factors.50 The level of albumin in the urine also showed an interaction between ADMA and kidney structures. Increased ACR indicates the occurrence of endothelial dysfunction, which can develop into hypertension.48\n\nSDMA and chronic kidney disease\n\nSDMA is an inactive stereoisomer that is produced concomitantly with ADMA.51 The synthesis of ADMA is affected by the reaction between superoxide anions and nitric oxide, which produces peroxynitrate, which results in tissue damage.52 SDMA is related to endothelial dysfunction and has a similar role to creatinine in the calculation of GFR. SDMA is excreted in the urine. SDMA, compared to ADMA, is more commonly found in patients with ESRD and is a more sensitive biomarker of alteration of renal function; therefore, SDMA can be treated as a specific biomarker.43 A study by Patel et al. (2019) also showed that SDMA is a stronger predictor for mortality risk in patients with CKD than ADMA.53 SDMA can stimulate ROS production, which induces proinflammatory effects in patients with CKD.54 In another study, it was found that in acute inflammation, ADMA but not SDMA is decreased. This became the initial question of whether SDMA and ADMA have different metabolic pathways and pathophysiological roles.51\n\nIn this study, the results showed that SDMA had a significant negative correlation with eGFR (p=0.00, r=-0.648; strong correlation) and a significant positive correlation with ACR (p=0.03, r=0.349; weak correlation). This can be interpreted as follows: the lower the eGFR is and the higher the ACR is, the more SDMA is detected. Another study also showed that there was an inverse correlation between SDMA and eGFR in patients with renal dysfunction; however, it was better at detecting CKD in early stages.55 SDMA also increases with creatinine in patients with CKD. Apparently, creatinine is less sensitive, and its detection is delayed56; therefore, SDMA has become a novel option to detect a decrease in GFR more accurately at earlier stages.57 Another study showed that there was an increase in SDMA at 6 hours after a 60% decrease in GFR, which reached the peak at 24 hours after nephrectomy and lasted for 7 days.57 In this study, we found a significant increase of SDMA level in late CKD stages and in dialysis patients (p=0.000 for both), showing that SDMA could predict CKD progression and be reduced by hemodialysis. However, how dialysis affects SDMA levels is not well known. Although initially SDMA was thought of no use, recent research shows that it has been a remarkable marker of renal function, with ESRD patients on dialysis showing the highest SDMA levels.58 We also found increased SDMA level in patients with DM (p=0.003). Elevated SDMA is associated with many diseases related with endothelial dysfunction, including DM. A possible explanation is the increased activity of arginine methyltransferase along with hyperglycemia, leading to increased SDMA synthesis.59,60\n\nThe limitation of this study was the lack of a healthy control group; therefore, the differences between the levels of the three oxidative stress markers in healthy people and patients with CKD are unknown. Another limitation was that we only collected samples from a single center, limiting the generalization of our results. In addition, some samples were CKD stage 5 with hemodialysis, in which the hemodialysis procedure itself could influence the data of these oxidative stress markers.\n\n\nConclusions\n\nIn patients with CKD in Indonesia, the oxidative stress markers 8-OHdG, SDMA, and ADMA correlated with the levels of eGFR and ACR, representing the level of severity of CKD. There were also significantly difference in 8-OHdG, SDMA, and ADMA levels among CKD stages, between dialysis and non-dialysis, DM and non-DM patients. In conclusion, 8-OHdG, ADMA, and SDMA levels can be used as prognostic markers and are potential candidates for future therapies in patients with CKD.\n\n\nData availability\n\nHarvard Dataverse: The role of oxidative stress markers in Indonesian Chronic Kidney Disease (CKD) patients https://doi.org/10.7910/DVN/ATSYBZ.61\n\nThe project contains the following underlying data:\n\n- Riset Cardiorenal 56.tab (raw per subject data)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nWritten informed consent for publication of the participants’ details was obtained from the participants.", "appendix": "References\n\nBikbov B, Purcell C, Levey A, et al.: Global, regional, and national burden of chronic kidney disease, 1990 – 2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2020; 395: 709–733. 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Med. 2014; 46(3): 244–252.\n\nValavanidis A, Vlachogianni T, Fiotakis C: (8-OHdG): A Critical Biomarker of Oxidative Stress and Carcinogenesis. J. Environ. Sci. Heal. Part C. 2009; 27(2): 120–139.\n\nNishikawa T, Sasahara T, Kiritoshi S, et al.: Evaluation of Urinary 8-Hydroxydeoxy- Guanosine as a Novel Biomarker of Macrovascular Complications in Type 2 Diabetes. Diabetes Care. 2003; 26(5): 1507–1512. Publisher Full Text\n\nSanchez M, Roussel R, Hadjadj S, et al.: Plasma concentrations of 8-hydroxy-2 ′-deoxyguanosine and risk of kidney disease and death in individuals with type 1 diabetes. Diabetologia. 2017; 1–8.\n\nAvin KG, Chen NX, Organ JM, et al.: Skeletal Muscle Regeneration and Oxidative Stress Are Altered in Chronic Kidney Disease. PLoS One. 2016; 11(8): 1–15. Publisher Full Text\n\nJacobson MH, Liu M, Wu Y, et al.: Oxidant stress and renal function among children with chronic kidney disease: a repeated measures study. Sci. Rep. 2020; 10: 1–10.\n\nDincer Y, Sekercioglu N, Pekpak M, et al.: Assessment of DNA Oxidation and Antioxidant Activity in Hypertensive Patients with Chronic Kidney Disease. Ren. Fail. 2008; 30: 1006–1011. PubMed Abstract \| Publisher Full Text\n\nAlsagaff MY, Pikir BS, Thaha M, et al.: Correlations between Total Antioxidant Capacity and 8-Hydroxydeoxyguanosine with Carotid-Femoral Pulse Wave Velocity in Chronic Kidney Disease. Indones Biomed. J. 2020; 12(3): 267–274. Publisher Full Text\n\nNavarro-García JA, Rodríguez-Sánchez E, Aceves-Ripoll J, et al.: Oxidative status before and after renal replacement therapy: Differences between conventional high flux hemodialysis and on-line hemodiafiltration. Nutrients. 2019; 11(11): 1–13. Publisher Full Text\n\nLiu X, Gan W, Zou Y, et al.: Elevated levels of urinary markers of oxidative DNA and RNA damage in type 2 diabetes with complications. Oxidative Med. Cell. Longev. 2016; 2016: 1–7. PubMed Abstract \| Publisher Full Text\n\nUeda S, Yuriko SY, Matsumoto Y: Asymmetric dimethylarginine (ADMA) is a novel emerging risk factor for cardiovascular disease and the development of renal injury in chronic kidney disease. Clin. Exp. Nephrol. 2007; 11(2): 115–121. PubMed Abstract \| Publisher Full Text\n\nMihout F, Shweke N, Bige N, et al.: Asymmetric dimethylarginine (ADMA) induces chronic kidney disease through a mechanism involving collagen and TGF- β 1. J. Pathol. 223(September 2010): 37–45. Publisher Full Text\n\nTsikas D, Bollenbach A, Hanff E, et al.: Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and homoarginine (hArg): the ADMA, SDMA and hArg paradoxes. Cardiovasc. Diabetol. 2018; 17(1): 1–4. PubMed Abstract \| Publisher Full Text\n\nCarlström M: Nitric oxide signalling in kidney regulation and cardiometabolic health. Nat. Rev. Nephrol. 2021; 17(9): 575–590. PubMed Abstract \| Publisher Full Text\n\nAlsagaff MY, Thaha M, Aminuddin M, et al.: Asymmetric Dimethylarginine: a Novel Cardiovascular Risk Factor in End-stage Renal Disease. J. Int. Med. Res. 2012; 40: 340–349. PubMed Abstract \| Publisher Full Text\n\nChoi HR, Lee SW, Jeon D, et al.: Association between estimated glomerular filtration rate (eGFR) and asymmetric dimethylarginine (ADMA) concentrations among the elderly in a rural community: a cross-sectional study. BMC Geriatr. 2019; 19(370): 1–9. Publisher Full Text\n\nFliser D, Kronenberg F, Kielstein JT, et al.: Asymmetric Dimethylarginine and Progression of Chronic Kidney Disease: The Mild to Moderate Kidney Disease Study. J. Am. Soc. Nephrol. 2005 Aug; 16(8): 2456–2461. PubMed Abstract \| Publisher Full Text\n\nAsmarawati TP, Thaha M, Mardiana N, et al.: Comparison of Asymmetric Dimethylarginine Levels Between Stages Three, Four, and Five Non-dialysis of Chronic Kidney Disease. Acta Medica Indones. - Indones. J. Intern. Med. 2016; 48(1): 28–34.\n\nOliva-damaso E, Oliva-damaso N, Rodriguez-esparragon F, et al.: Asymmetric (ADMA) and Symmetric (SDMA) Dimethylarginines in Chronic Kidney Disease: A Clinical Approach. Int. J. Mol. Sci. 2019; 20(3668): 1–15.\n\nVallance P, Leone A, Calver A, et al.: Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet (London, England). 1992 Mar; 339(8793): 572–575. Publisher Full Text\n\nWang J, Siew A, Li X, et al.: Relations between plasma asymmetric dimethylarginine (ADMA) and risk factors for coronary disease. Atherosclerosis. 2006; 184: 383–388. PubMed Abstract \| Publisher Full Text\n\nYusuf M, Thaha M, Yogiarto RM, et al.: Dimethylarginine Dimethylaminohydrolase 2 Gene Polymorphism and Its Association with Asymmetrical Dimethyl Arginine in Hemodialyzed Patients *. Open J. Nephrol. 2013; 03: 75–81. Publisher Full Text\n\nSciacqua A, Grillo N, Quero M, et al.: Asymmetric dimethylarginine plasma levels and endothelial function in newly diagnosed Type 2 diabetic patients. Int. J. Mol. Sci. 2012; 13(11): 13804–13815. PubMed Abstract \| Publisher Full Text\n\nTsioufis C, Dimitriadis K, Antoniadis D: Inter-Relationships of Microalbuminuria with the Other Surrogates of the Atherosclerotic Cardiovascular Disease in Hypertensive Subjects. Am. J. Hypertens. 2004; 17: 470–476. PubMed Abstract \| Publisher Full Text\n\nKielstein JT, Donnerstag F, Gasper S, et al.: ADMA Increases Arterial Stiffness and Decreases Cerebral Blood Flow in Humans. Stroke. 2006; 37: 2024–2029. PubMed Abstract \| Publisher Full Text\n\nTsioufis C, Dimitriadis K, Andrikou E, et al.: Hypertension: A Cross-sectional Study. Am. J. Kidney Dis. 2010; 55(6): 1050–1059. PubMed Abstract \| Publisher Full Text\n\nMemon L, Spasojevic-kalimanovska V, Bogavac-stanojevic N, et al.: Assessment of Endothelial Dysfunction: The Role of Symmetrical Dimethylarginine and Proinflammatory Markers in Chronic Kidney Disease and Renal Transplant Recipients. Dis. Markers. 2013; 35(3): 173–180. PubMed Abstract \| Publisher Full Text\n\nCavalca V, Veglia F, Squellerio I, et al.: Circulating Levels of Dimethylarginines, Chronic Kidney Disease and Long-Term Clinical Outcome in Non-ST- Elevation Myocardial Infarction. PLoS One. 2012; 7(11): 1–8. Publisher Full Text\n\nPatel L, Kilbride HS, Stevens PE, et al.: Symmetric dimethylarginine is a stronger predictor of mortality risk than asymmetric dimethylarginine among older people with kidney disease. Ann. Clin. Biochem. 2019; 56(3): 367–374. PubMed Abstract \| Publisher Full Text\n\nSchepers E, Glorieux G, Dhondt A, et al.: Role of symmetric dimethylarginine in vascular damage by increasing ROS via store-operated calcium influx in monocytes. Nephrol. Dial. Transplant. 2009; 24: 1429–1435. PubMed Abstract \| Publisher Full Text\n\nWasilewska A, Taranta-janusz K, Zoch-zwierz W, et al.: Is plasma symmetric dimethylarginine a suitable marker of renal function in children and adolescents ?. Scand. J. Urol. Nephrol. 2012; 46: 58–64. PubMed Abstract \| Publisher Full Text\n\nBode-Böger SM, Scalera F, Kielstein JT, et al.: Symmetrical dimethylarginine: A new combined parameter for renal function and extent of coronary artery disease. J. Am. Soc. Nephrol. 2006; 17(4): 1128–1134. PubMed Abstract \| Publisher Full Text\n\nKielstein JT, Veldink H, Martens-lobenhoffer J, et al.: SDMA is an early marker of change in GFR after living-related kidney donation. Nephrol. Dial. Transplant. 2010; 26: 324–328.\n\nOliva-Damaso E, Oliva-Damaso N, Rodriguez-Esparragon F, et al.: Asymmetric (ADMA) and symmetric (SDMA) dimethylarginines in chronic kidney disease: A clinical approach. Int. J. Mol. Sci. 2019; 20(15). PubMed Abstract \| Publisher Full Text\n\nDayir BS, Hamzah MI, Khudair MSH: Serum levels of Asymmetric dimethyl arginine and Nitric oxide in patients with prediabetes and type2 diabetes mellitus. Ann. Trop. Med. Public Heal. 2020; 23(2): 11–16. Publisher Full Text\n\nTsikas D, Bollenbach A, Hanff E, et al.: Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and homoarginine (hArg): The ADMA, SDMA and hArg paradoxes. Cardiovasc. Diabetol. 2018; 17(1): 1–4. PubMed Abstract \| Publisher Full Text\n\nAlsagaff MY, Thaha M, Pikir BS, et al.: The role of oxidative stress markers in Indonesian Chronic Kidney Disease (CKD) patients.2021. Harvard Dataverse, V1, UNF:6: ADR9/7LlZHDjbs18g5Wrgw== [fileUNF].Publisher Full Text" }	[ { "id": "227800", "date": "07 Feb 2024", "name": "Dorota Formanowicz", "expertise": [ "Reviewer Expertise medical doctor", "internal medicine specialist", "conducting research studies in CKD patients", "oxidative stress", "inflammation", "chronic disorders" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is an interesting article, certainly in the context of the studied group, as there are few studies from the Indonesian region, but it has some shortcomings. What is the goal? It is not specified; please complete it. The conclusions are a description of the results obtained in the study and not conclusions based on these results.\nI was wondering about the title. Is the Indonesian group so unique that it should be mentioned in the title? If yes, it should be underlined in the text.\nI was wondering whether analyses taking into account CKD1 and CKD2 are justified. There were only 2 and 4 people in these groups, respectively - individual variability may affect the results obtained, and they do not necessarily reflect the entire group. Maybe researchers should combine groups 1 and 2 and label them CKD1-2.\nThe researchers presented a correlation of eGFR with the assessed parameters of oxidative stress, but I have a question: what was the eGFR in individual groups? Moreover, I have a question about whether this correlation was maintained for every group or only for the group of dialysis patients. I didn't find it in the group characteristics; a similar question concerns ACR: what was it like in individual research groups?\nThere is an error in the table regarding the characteristics - it should be BMI [kg/m2] and not cm/m2\nAnother question - if the history of dialysis was taken into account in the context of correlation studies with oxidative stress parameters, I have a question about how long the patients were on dialysis. Who was considered a dialysis patient - everyone who was currently on dialysis or those who, for example, had been on dialysis for 06 months? I did not find inclusion criteria for specific groups.\nThe results of lipid metabolism tests are described, but I have not found any information on treatment. Are these the results for people treated with, e.g., statins?\nA missing unit in the text for BMI is a sentence: \"with an average body mass index (BMI) of 26.08±4.65 ...\" - this should be completed.\nI have a question: when exactly were blood samples taken - especially from hemodialysis patients - standardly, samples are taken before dialysis (2nd dialysis session per week)? In this study, this does not seem to have been maintained, and there are different downloads - which influences the results.\nLimitations of the study - the most significant limitation is the number of studied patients. The groups are too small, especially CKD 1i and CKD 2. Did the authors check the minimum group size so that the results obtained could be considered reliable?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-132
https://f1000research.com/articles/10-1023/v1	08 Oct 21	{ "type": "Research Article", "title": "Student biocuration projects as a learning environment", "authors": [ "Katherine E. Thurlow", "Ruth C. Lovering", "Sandra De Miranda Pinheiro", "Katherine E. Thurlow", "Sandra De Miranda Pinheiro" ], "abstract": "Background: Bioinformatics is becoming an essential tool for the majority of biological and biomedical researchers. Although bioinformatics data is exploited by academic and industrial researchers, limited focus is on teaching this area to undergraduates, postgraduates and senior scientists. Many scientists are developing their own expertise without formal training and often without appreciating the source of the data they are reliant upon. Some universities do provide courses on a variety of bioinformatics resources and tools, a few also provide biocuration projects, during which students submit data to annotation resources. Methods: To assess the usefulness and enjoyability of annotation projects a survey was sent to University College London (UCL) students who have undertaken Gene Ontology biocuration projects. Results: Analysis of survey responses suggest that these projects provide students with an opportunity not only to learn about bioinformatics resources but also to improve their literature analysis, presentation and writing skills. Conclusion: Biocuration student projects provide valuable annotations as well as enabling students to develop a variety of skills relevant to their future careers. It is also hoped that, as future scientists, these students will critically assess their own manuscripts and ensure that these are written with the biocurators of the future in mind.", "keywords": [ "Biocuration", "Gene Ontology", "community curation", "student gene annotation" ], "content": "Introduction\n\nBioinformatics is utilised in a wide range of scientific disciplines, including data science and statistics as well as the biological sciences.1 As such, a research project in bioinformatics requires the incorporation of skills from a multitude of areas and can be utilised in a plethora of future careers, whether specifically based on computational biology or otherwise. Recent technological advancements in high-throughput research have resulted in vast amounts of data output, which is outstripping analysis capabilities.2 Therefore, a priority for bioinformatics research is to increase efficiency and the rate at which peer-reviewed experimental data is captured.\n\nBiocuration is the process of capturing biological knowledge in a database, usually through the creation of annotations. Typically, this process involves an in-depth review of the published literature and then, using standardised terms and identifiers, condensing the information into a computer-readable format. This process has many similarities to a literature review project that many students undertake during their undergraduate and postgraduate studies. The main difference being that at the end of a literature review the student must provide a well-written summary of their investigations, whereas a biocurator needs to learn and apply knowledgebase specific annotation rules to summarise the reviewed data.\n\nSince the turn of the century and launch of the postgenomic era it has become more apparent that bioinformatics has the potential to improve the research efficiency of biologists, biomedical researchers, statisticians and clinicians alike. With the wide range of bioinformatics resources to choose from, finding and using the gold standard resources can be challenging. Consequently, many universities now offer bioinformatics courses to undergraduates, postgraduates and staff. While many of these courses focus on providing training on specific resources, such as Ensembl,3 UniProt4 or functional analysis tools,5-7 a few universities, including University College London (UCL), encourage students to contribute annotations to public biological knowledgebases.8-12 For example, over the past 10 years the Functional Gene Annotation group (UCL) has provided a 10-week bioinformatics MSc module as well as a Gene Ontology biocuration project to undergraduate and postgraduate students.\n\nThe aim of the Gene Ontology (GO) is to capture the vast amount of published biological and biomedical data describing gene products into an accessible and computer-readable format.13,14 The GO knowledgebase is split into two primary intertwining sections. Firstly, the ontology provides summary statements (GO terms) that describe the molecular functions (e.g. enzyme activity), biological processes (e.g. immune response) and cellular locations (e.g. cytoplasm) of gene products, with hierarchical and directional relationships between GO terms (Figure 1). Secondly, GO annotations link specific gene or gene product identifiers to the ontology terms, thereby placing the gene products within a biological context and allowing a statement to be made about their functional role and location in the cell. The standardisation of language through GO terms supports accurate curation. A variety of approaches are taken to create GO annotations, ranging from manual curation, by biocurators, based on information presented in peer-reviewed published literature (Table 1), through to automatic pipelines, extracting data from bioinformatics databases, such as InterPro and Reactome.15-17 There is an additional novel, third aspect to GO; GO-Causal Activity Models (GO-CAMs), which compiles GO annotations into comprehensive gene function models and can include causal inferences between molecular activities.18 This feature of GO was not available for any of the projects within this study.\n\nA fragment of GO, representing the relationships between the GO term ‘heart development’ (highlighted in yellow) and its parent, less specific GO terms. Graph downloaded from QuickGO,44 black arrows indicate is_a relations between terms and blue arrows indicate part_of relations (https://www.ebi.ac.uk/QuickGO/term/GO:0007507) Access date: 19/05/2021.\n\nTwo annotations, created during a UCL GO biocuration project, associating human miR-153b-3p with molecular function and biological process GO terms. These annotations were supported by a luciferase assay45 and, therefore, assigned a direct assay evidence code.14 The annotation extension field enables the biocurator to capture the miRNA target, APP (UniProtKB ID: P05067) and the tissue (brain) in which these activities and processes are occuring in. Data downloaded from QuickGO,44 access date: 21/05/2021.\n\nThe GO has been cited by over 100,000 publications as of 2020,18 demonstrating its value and global use. The increasing quantity of high-throughput data analysis being conducted in recent years has meant annotation of genomes and proteomes is of particular importance, as the quality of gene annotations impacts on the accuracy of data interpretation. The GO is being used in Genome Wide Association Studies, to increase the likelihood of identifying disease-risk genes through the grouping of genes involved in the same disease associated pathway.19,20 Furthermore, GO is used for biomarker identification,21 the identification and further elucidation of risk predictions,22 as well as to interpret transcriptomic and proteomic datasets associated with systemic diseases affecting multiple systems, such as cancers.23-25 Thus, current research projects are becoming more reliant on computationally accessible GO annotation data to identify gene products that could be used as either potential drug targets, or as prognostic or diagnostic biomarkers. GO annotations extensions26 also allow for elucidation of cellular and tissue context for gene function or involvement in particular processes (Table 1), which can also inform research with drug repurposing or assessment of off-target effects.\n\nThe interpretation of high-throughput datasets can be hampered by the researcher’s lack of understanding of the underlying resources used in the analysis. Therefore, increasing education in bioinformatics will provide researchers the knowledge and skills to appropriately interpret their data. Provision of sufficient bioinformatics training has long been a challenge,27-29 which is being partially addressed globally with 62 events on the TeSS Bioinformatics training platform30 (access date: 02/06/21) and over 100 open-source training materials and resources for bioinformatics available from the GOBLET training portal31 (access date: 02/06/21), many of which are available directly from the resource providers. However, while the TeSS platform includes events covering data mining and machine learning, only a handful of these events are specifically for biologists and clinicians using bioinformatics. Furthermore, there is a lack of biocurator focused courses.27-29\n\nThe primary objective for a UCL student undertaking a GO biocuration project is to annotate within a theme to complete a thesis. Consequently, these projects involve in depth reading of scientific articles surrounding a disease-related process or set of genes. For example, recent project titles have focused on curating microRNA regulation of junctional proteins at the blood-brain barrier and amyloid processing in the context of Alzheimer’s disease, as well as the role of specific signalling pathways in heart development (https://www.ucl.ac.uk/cardiovascular/student-projects). The GO annotations created by the students are checked by a professional biocurator and submitted to the GO Consortium knowledgebase. As the Functional Gene Annotation group has been previously funded by the British Heart Foundation, Alzheimer’s Research UK and Parkinson’s UK, the projects have primarily surrounded cardiovascular and neurological disease-associated processes or gene sets.\n\nThere are three primary overarching learning objectives for a GO biocuration project, firstly, to improve the student’s understanding of experimental methods through critical analysis of articles identified through a literature search. Secondly, for the student to gain extensive experience of using bioinformatics resources in concert, applying theoretical knowledge to practical utilization. Finally, to increase the student’s understanding of ontologies and the process of manual annotation. Secondary learning outcomes include improving the student’s knowledge surrounding the biological context of the project, gaining confidence in GO analysis, as well as a change in the student’s approach to both general research and bioinformatics research in particular. Many of the projects carried out as part of one of the degrees represented in this study also require the student to complete a 10,000 word dissertation and to provide a 10-15 minute project presentation. For the purpose of this study, the skills students can expect to gain were split into those that would be acquired during the majority of UCL student projects, such as writing a dissertation and giving a project presentation, and skills specific to the GO biocuration projects, such as the process of curation and use of bioinformatics databases and tools (Figure 2).\n\nThe skills highlighted in blue are general project skills which are common to undergraduate and graduate dissertations. The skills highlighted in green are specific to the GO biocuration project. Although, some, or all, of these skills may also be gained in other types of research projects.\n\nStudents will typically curate around 15-30 articles per project, requiring a good understanding of experimental methods, in addition to strong literature review and critical analysis skills. Each project usually leads to the creation of between 200 to 500 annotations associated with 35 to 55 gene products. To date, over 5,000 GO annotations have been associated with almost 900 entities, based on the review of over 500 articles by UCL students. As such, these student projects can substantially increase the coverage of the GO, through providing new GO annotations. For example, one project provided more detailed annotations describing the role of TGFB1, BMP5, ENG and BMPR1A in heart development (Figure 3). In addition, the interactions captured through GO biocuration projects are included in the existing molecular interaction datasets32 and can help elucidate relationships between interacting gene products (Figure 4).\n\nThe histogram indicates the number of 'heart development' GO terms, associated with seventeen specific proteins, that existed before the start of the student’s project (orange) and contributed by the student (blue). This student’s project focused on the curation of proteins with a known role in heart development. At the end of the project the student had submitted over 500 GO annotations through the review of 25 articles and associated specific GO terms from the 'heart development' domain of the ontology with the prioritised proteins.\n\nThis network was created using Cytoscape7 and demonstrates the contribution of this project to an interaction network of blood-brain barrier-associated proteins and miRNAs.\n\nStudent GO biocuration projects have the potential to substantially increase a student’s skill-set, improving their ability to use bioinformatics resources to answer a research question, and increasing their understanding of experimental methods and ontologies. Thus, these students will be better placed to use biological and biomedical knowledgebases effectively in their future careers. Additionally, these projects have a secondary benefit to the wider biocuration community through a considerable contribution to the GO knowledgebase and the training of potential future expert biocurators and community biocurators. Another potential benefit, is that as future researchers these students may write future articles more sympathetically to biocuration, thus making data curation more efficient overall. This study aims to assess the usefulness and enjoyability of GO biocuration projects from the student perspective through analysis of quantitative and qualitative survey responses.\n\n\nMethods\n\nThe aim of this study was to consult past and present students to assess whether GO biocuration projects provide relevant and useful skills to students. A secondary research question addressed whether these projects lead to a wider appreciation of bioinformatics and its applications and also whether students would be interested in a community biocuration initiative.\n\nAn invitation to complete an open online questionnaire was circulated via email to 22 students who had either completed or were undertaking a GO biocuration project. In addition to specific questions about their views on the usefulness and enjoyability of various aspects of the project, demographic questions were collected. The first email inviting students to complete the survey was sent on 31st March 2021. Reminders were automatically sent to students that did not complete the survey on 7th, 14th and 20th April, and the survey closed on 7th May 2021.\n\nThe survey (Extended data: Table S1A46), created using opinio software (https://opinio.ucl.ac.uk; version 7.12), was initially based around the Kirkpatrick model for assessment of training,33 using three fundamental evaluation categories; reaction to training, learning outcomes and behaviour change, comprising 16 questions. Of these, 8 were considered demographic questions and 8 assessed the three evaluation categories. Quantitative metrics were obtained through the use of binary yes/no options or ratings ranging from 1-5. Comment boxes were included with several of the questions, to obtain qualitative data and supporting statements.\n\nDemographic questions captured the year of study, the course undertaken while completing the project, the current role of the participant, whether the participant had previously attended a 10-week bioinformatics module, the participants view of their bioinformatics knowledge before and after the project, how frequently they use (or expect to use) bioinformatics resources and why they chose the GO biocuration project.\n\nReaction to training was assessed by asking the participants to rank the aspects of the project they found most useful to their personal development and to rank the aspects of the project they found most enjoyable.\n\nLearning outcomes and behaviour change was investigated by asking the participants to rank the extent to which they agreed with five statements encompassing an improvement in overarching biological context knowledge, confidence in GO analysis interpretation, gaining unique skills and whether the way in which they conduct general and bioinformatics research had changed due to undertaking their project.\n\nIn addition, the participants were asked whether their views on the relevance/impact of bioinformatics had changed, whether they would recommend the project to future students and whether they would be interested in taking part in a community biocuration initiative.\n\nThe qualitative data was analysed using NVivo software version 12 and text condensation methodology,34 as well as two independent researchers providing an overall impression of the data. Briefly, text condensation comprised thematic analysis of all qualitative responses (i.e., comment box content) where themes were characterised according to the research questions and project aspects. “Meaning units”, i.e., particular statements made by respondents, were identified from the text and coded to a particular theme. Repetition allowed for refinement and substantiation of the coding and clarification of themes. Condensation of these meaning units allowed for summation of the overall impression for each theme identified (Underlying data: Table S946) and collation of concepts to support the quantitative findings of the survey.\n\nStatistical analysis was conducted primarily on the reaction to training questions, but also to ascertain significance on learning outcomes and demographic questions including prior and post bioinformatics knowledge. Using Microsoft Excel for Mac, version 16.52, an F-test was performed to determine variance between; positive vs non-positive responses and then negative vs non-negative responses as well as between prior and post project bioinformatics knowledge and bioinformatics module attendance (Extended data: Table S446). After determination of variance, t-tests were performed between the groups to ascertain significant difference (Extended data: Table S546). Lastly, Chi2 tests were performed for all project aspects and learning outcomes to ascertain the significance of observed percentage responses compared to expected percentage responses (Extended data: Table S646). The expected percentage response for each rating 1-5 was 20%. ANOVA comparison of means was carried out to ascertain whether there was any correlation between attendance of the Genetics of Human Disease bioinformatics module and prior/post project bioinformatics knowledge (Extended data: Table S746).\n\nFollowing UCL ethical guidelines, the research was classed as exempt from UCL ethical approval because the research involved the use of a non-sensitive, completely anonymous survey, the participants are not defined as “vulnerable”, and participation would not induce undue psychological stress or anxiety. Participants were informed in the invitation email and at the start of the survey that the submitted responses would be kept anonymous but that the data collected would be made public and that the feedback would be used to revise future annotation projects. Consent to participate in the study was implied by completion of the questionnaire. In addition, this article provides no information which would enable the answers to the survey to be linked to specific participant identities.\n\n\nResults\n\nThe survey examined the respondents view of their biocuration project with respect to a variety of aspects using both qualitative and quantitative questions. For the purpose of analysis, the questions were split up into demographic style questions and questions relevant to the biocuration project.\n\nThe survey was emailed to 22 past and present students who had undertaken GO biocuration projects with the Functional Gene Annotation group at UCL, with an 82% response rate and a 73% completion rate (Underlying data: Table S1B46). The demographic questions collected data on: the year the project was completed, stage of study, current job role, attendance of a 10-week UCL MSc bioinformatics module and current or expected frequency of use of bioinformatics resources (Underlying data: Table S246). Questions about the participants' view of their prior and post project bioinformatics knowledge and the open question of why respondents chose a biocuration project were also included.\n\nThe majority (n = 13) of the respondents embarked on a biocuration project as part of an MSc qualification. Of the remaining respondents, three were iBSc students and two were PhD students. The survey cohort completed their projects over a span of ten years (2011-2021) with the majority of respondents completing their projects in 2019 or later (n = 11). The 18 respondents had a wide variety of current roles, including medical writer, clinical geneticist and post-doctoral fellow (Table S2). This provided an insight into how useful and relevant a GO biocuration project could be to areas of science that are not directly focused on bioinformatics or annotation, with one respondent stating: “I think bioinformatics is an essential skill to have and it can really help you with other areas too” (respondent ID: 3440328).\n\nOver three quarters (77.8%) of respondents attended the 10-week UCL bioinformatics module, but this did not seem to correlate with either prior (t = 0.62, p-value = 0.27) or post (t = 1.34, p-value = 0.10) project bioinformatics knowledge (Table S5). Although those that did attend the module had higher average scores of both prior (attended = 2.43, did not attend = 2.00) and post (attended = 4.17, did not attend = 3.67) bioinformatics knowledge, this difference was not statistically significant (p-value = 0.54 and 0.20 respectively from ANOVA comparison of means, Table S7). Almost all students (89.47%) stated that their understanding of bioinformatics was average or below at the start of their projects, with two respondents rating both their prior and post knowledge as ‘excellent’ (Figure 5). Both of these individuals chose a biocuration project in order to broaden their skill-set and learn how to apply this knowledge to research, “I was keen to develop my skills in this area as I have always been interested in bioinformatics. I also wished to be involved in opportunities to expand this knowledge to real applications” (3445442). Overall, respondents reported a significant improvement in bioinformatics knowledge after their project compared to before their project (Figure 5, t = −5.43, p-value = 5.43 × 10−6) with 87.50% rating themselves above average (i.e. good or excellent).\n\nThe survey included two questions to investigate whether the student’s view of their own bioinformatics knowledge had changed after undertaking a biocuration project. Respondents rated the bioinformatics knowledge they had acquired before the start of the project (prior project knowledge level) and at the end of the project (post project knowledge level) using ratings ranging from non-existent to excellent.\n\nThe majority of respondents (73.3%) either currently use, or expect to use, bioinformatics resources on a regular basis (Figure 6). One respondent (3445928) stated: “I have used the resources a lot since. Also a lot of my PhD peers have never used these resources and always say how they wish they knew how to”. Only one participant identified that they do not expect to use bioinformatics resources in the future, this individual is a current student and had to change their project plans due to COVID restrictions. Their response suggests that this student may be planning to follow either a laboratory-focused or non-research career path. The two ‘a few times a year’ responses were from a student and a medical writer, with the clinical geneticist, post-doctoral fellow and all of the PhD students, responding ‘multiple times a week’. All of the ‘everyday’ responses were from biocurators. The overall impression from our results is that the skills obtained through this project are highly transferable and that bioinformatics resources are relevant to a multitude of careers.\n\nRespondents were asked how frequently they use (or expect to use for current students) bioinformatics resources after completion of their project.\n\nThere were a variety of reasons students initially chose a biocuration project, including the recent COVID pandemic restricting availability of laboratory-based projects and the overarching biological focus of the project e.g. Alzheimer’s Disease (Table S1B, Question 6). However, the three most common reasons were:\n\n1. To gain new skills that would be useful in the future, “because I wanted to learn new skills and challenge myself. Before the project, I only had little experience with bioinformatics” (3440328).\n\n2. A preference for dry lab or computational work, “I was interested in improving my understanding of genetics, data science and paper interpretation as well as undertaking a non-lab based project” (3440130).\n\n3. Personal interest in bioinformatics, “It was an area that I wanted to explore as a potential career path” (3445942).\n\nThe encompassing impression is that the majority of students undertook this project because they knew (or had been told) it would be useful, but not necessarily understanding why.\n\nQuantitative analysis of both enjoyability and usefulness of project aspects as defined by the authors was carried out to assess the three primary learning outcomes of the projects. The aspects were split into general skills, such as carrying out a literature search, that could be expected to be gained by undertaking any research project that included a final dissertation and presentation, and specific bioinformatics skills, unique to a GO biocuration project, such as learning to use ontologies (Underlying data: Table S346). Respondents rated each aspect of their biocuration project from not at all useful or enjoyable to extremely useful or enjoyable. All responses were statistically significant with Chi2 p-values ranging from 5.19 ×10−9 for enjoyability of presentation to 7.61 ×10−49 for usefulness of both critical evaluation of articles and write up (Table S6). The majority of our respondents (12 out of 18) did not report any negative experiences, and this view is supported by statements such as: “(A) bioinformatics project is a good way to learn organizing information with a clear order and map their interactions.” (3440087, Table S1B, Question 6). There were some negative responses (Underlying data: Table S846), with one participant stating that Cytoscape was ‘not at all’ useful or enjoyable and another that literature searches were not enjoyable. Presentations were considered by two participants as only slightly useful and by two as only slightly enjoyable. Only one participant gave a low score on several aspects. The student submitting the highest number of negative responses for this section considered GO annotation, databases, BLAT, BLAST and Cytoscape as ‘slightly useful’. As this respondent didn’t provide any comments from which we could have understood why they answered negatively for these particular aspects, but they did score enjoyability of these aspects higher.\n\nUsefulness and enjoyability of general skills\n\nA significant proportion (78.1%; t = 10.15; p-value = 3.9 × 10−8) of responses were positive about the general skills and tasks, either selecting considerably or extremely useful or enjoyable, and almost all responses (95.3%; t = 32.72; p-value = 6.3 × 10−15) were rated “non-negative” when including the neutral option “moderately”. The overall positive response scores for usefulness (96.7% non-negative) were higher than for enjoyability (93.3% non-negative).\n\nLike other student biocuration projects, the UCL student projects involved extensive searching of literature and critical evaluation of research, “It provided a set of tools that could be used as part of a literature search on an area of interest and an effective way to search for experimental data on a specific set of genes.” (3445942), both of which will be vital skills for virtually any subsequent scientific career.21 The survey confirmed that the majority of respondents (88%) particularly valued the critical evaluation skills they developed during their projects. The comments provided on this aspect of the biocuration project support this (Table S1B, Question 7, other specify), a typical response was “this is something that is very useful not only for a career in scientific curation but many other scientific career paths” (3440233). Almost all of the respondents considered that writing their dissertation was the most useful general aspects of the biocuration projects, with 95% rating this skill as considerably or extremely useful (Figure 7). Fewer respondents indicated that the literature search aspect of the project provided them with useful skills (70% positive responses) although 100% of responses were non-negative.\n\nThe survey examined how the respondents ranked the usefulness of the skills they had gained through literature searching (lit. search), writing their dissertation (writing), presenting their project (presenting) and critically evaluating published articles (evaluation). In addition, the survey examined how much the respondents enjoyed these tasks. The ranking options range from “not at all” useful/enjoyable to “extremely” useful/enjoyable.\n\nThis general agreement to the usefulness of these general skills, which would also have been developed during a laboratory-based project, also aligns with the qualitative findings. Over 30% of respondents identified critically analysing experimental data and literature searching as highly relevant skills to their current roles, with respondent 3440337 stating “There was a lot of searching literature and critical evaluation of research, both of which will be vital skills for virtually any subsequent career move in this field.” Furthermore, respondent 3440130 mentioned “Improving my ability to critically evaluate papers was crucial in my personal development as it allowed me to widen my understanding of different topics” (Table S1B, Question 9). In general, the responses to the level of enjoyment of these tasks were similar to the views on the usefulness of these skills (Figure 7) with only a 3.4% difference in non-negative responses.\n\nUsefulness and enjoyability of biocuration specific skills\n\nThe positive response score for usefulness (80%) was only slightly lower than for enjoyability (81.3%), with the overall biocuration specific aspects having a high rate of positive responses (80.6%; t = 19.62; p-value = 9.90 × 10−16). However, almost all responses (95.8%; t = 43.38; p-value = 4.14 × 10−23) were “non-negative”, when including the neutral option “moderately (Figure 8).\n\nThe survey examined how the respondents ranked the usefulness of understanding about and using ontologies and GO specifically, creating GO annotations, using a variety of databases and bioinformatics resources (Cytoscape, BLAT and BLAST). In addition, the survey examined how much the respondents enjoyed using these resources and creating GO annotations. The ranking options range from “not at all” useful/enjoyable to “extremely” useful/enjoyable.\n\nWhen all responses concerning the usefulness of a biocuration project are compared, learning how to effectively use bioinformatics databases was ranked third most useful, after writing dissertation and critical evaluation of articles (Table S1B, Question 7). This was supported by respondents’ statements such as “I felt I learned a great deal … from learning about the existence of many different databases, to what they offered and how to use them and why” (3432165). As expected, due to the wide range of current job roles of the respondents (Table S2), the positive responses for usefulness were slightly lower for the biocuration specific aspects of the project compared to the general aspects, with a difference of 4.6%. However positive responses for enjoyability were 8.2% higher for biocuration specific aspects than for the general aspects.\n\nAlthough understanding ontologies may be regarded as a bioinformatics skill relevant to a broader range of scientific careers than GO annotation, GO annotation was found to be just as useful as learning about ontologies. One comment “Through both background literature searches and the annotation process of the project I came to realise how influential and valuable ontologies and annotations can be” (3436438), suggests that the similar rating of GO annotation and ontologies is because the two are inextricably linked and therefore the process of annotation aids in the general understanding and appreciation of ontologies and their applications. Although the usefulness ratings were the same for GO and general ontologies, people generally enjoyed using GO more, suggesting the interface of GO is more user friendly, or that the frequent use of the GO browsers made these more enjoyable tasks than occasionally looking for other ontology terms. Alternatively, a student’s rating of the GO ontology as enjoyable may reflect the satisfaction of being able to see their annotations in the GO knowledgebase. The statement “It was really satisfying to see my annotations get uploaded onto the databases” (3440328) supports this idea (Table S1B, Question 9). Learning how to use the bioinformatics resources BLAT, BLAST and Cytoscape were generally found to be the least useful aspects of the projects.\n\nFive learning outcomes were investigated: improved knowledge of the biological context of the project; unique skills gained; increased confidence in interpretation of GO analyses; altered approach to both general and bioinformatics research. Almost all responses (98.6%) either somewhat or strongly agreed that they had achieved these learning outcomes (Figure 9). Responses were statistically significant, with Chi2 values ranging from 6.0 × 10−26 for confidence in GO analysis to 7.63 × 10−72 for altered approach to bioinformatics research (Table S6). All but one of the respondents (93.3%) strongly agreed that their appreciation of bioinformatics and biocuration increased after undertaking an annotation project (Figure 9), with one respondent (3446071) stating “I never knew how important bioinformatics was until after this project” (Table S1B, Question 12). The individual who answered ‘somewhat agree’ (3432165) to this question also stated that “Having acquired some knowledge about bioinformatics through the project, I feel I might be able to use it more efficiently and effectively in future research projects”, which suggests their response may be down to a lack of confidence rather than a lack of skills, supported by the fact they also answered ‘somewhat agree’ in their confidence in GO analysis. The majority of respondents (78.6%) strongly agreed that their conduction of general scientific research was also changed as a result of their projects (p = 5.88 × 10−49) with one student stating “… any future research I conduct will ALWAYS state very clearly the species of all entities utilised!” (3436438). The submitted statements suggest this behaviour change was due to a greater appreciation of bioinformatics by the students: “This project definitely helped me have a clearer understanding of what bioinformatics is and the plentiful tools available. This made me appreciate bioinformatics more and the potential and power it has” (3440328), “I never knew how important bioinformatics was until after this project” (3446071, Table S1B, Question 12).\n\nFive questions were included in the survey to assess whether a biocuration project provided the appropriate environment to meet the learning outcome aims. Respondents were asked whether they strongly disagree, somewhat disagree, neither agree nor disagree, somewhat agree or strongly agree that they had achieved each of the learning outcomes of the project.\n\nAs only 18 students completed the survey there was a limited amount of information that could be gained from the answers provided in the comment fields. However, the respondents’ statements provided a clear consensus about the value of these projects. In addition, these responses covered a very broad range of issues, which provided some interesting insights about the respondents’ view of their annotation projects (Table S9). Only one respondent did not provide any accompanying comments (Table S1B).\n\nKey themes were identified in the student responses provided in all the available comment boxes and coded phrases or “meaning units” were assigned to each theme (Table 2). The meaning units were then summarised using the text condensation methodology34 (Table S9). The theme with the highest number of meaning units was ‘relevant new skills’ (n = 31) followed by appreciation (n = 19). Each theme had meaning units coded to it from multiple participants, with a range of 29.4% of participants mentioning effective use of databases and project themes to 71% of participants mentioning relevant new skills and newfound appreciation of bioinformatics (Table 2).\n\nComments provided in response to the survey (Underlying data: Table S946) were combined to identify key themes. Meaning units are extracted phrases from the survey responses. These meaning units were coded to themes identified through initial text analysis of all qualitative responses. The number of respondents whose comments were coded to each theme is shown in brackets.\n\nAn unexpected finding was that almost all of our participants stated they would be interested in participating in a community biocuration initiative (Table S1B, Question 15). Motivation was to both continue implementing their acquired biocuration skills (“I think it is important to continue to work on my skills I have gained so far, so being able to be involved in opportunities like these would be very beneficial”, 3445442) and to contribute to the GO (“I believe community biocuration initiative can promote biocuration and in turn aid in improving under represented areas in the ontology. At the same, the community biocuration initiative can aid the incorporation of biocuration in university/research institution settings”, 3445942).\n\n\nDiscussion\n\nThis survey of students that had either completed or were currently undertaking a GO biocuration project confirmed the value of these assignments. The primary research question, whether GO biocuration projects provide useful and relevant skills to students, was overwhelmingly positive. Additionally, the majority of tasks were considered to be enjoyable as well as useful. The secondary research question addressed whether GO biocuration projects provide a greater overall appreciation and understanding of bioinformatics applications. This was confirmed to be the case, with the majority of participants (80%) confirming that their views of bioinformatics had changed.\n\nThe number of respondents to our survey (n = 18), limits the interpretation of this data, however the responses are in agreement with those of other similar studies investigating the usefulness of biocuration student projects.11 Hosmani et al. (2019),11 discuss the benefits to students undertaking manual annotation efforts including the development of a better understanding of genomics data, the retention of the students in the sciences, and their inclusion on peer-reviewed publications. Our study did not specifically investigate student retention, however all of the respondents to date are still associated with a scientific or medical role.\n\nWhile it is clear that undertaking a biocuration project leads to the acquisition of general skills that are transferable to other areas of science10,11 all of our survey respondents appreciated the future benefit of using bioinformatics resources in a research project setting, with one student explaining that “The experience was also useful for making me aware of future possibilities in terms of where I could apply this knowledge” (3445442). Manual gene annotation involves more than simply copying and pasting information from one resource to another.10 The biocurator has to learn how to use a variety of bioinformatics resources, understand how to recognise the appropriate gene identifiers, as synonyms are often included in articles rather than approved symbols, and understand how to select the appropriate ontology term for the data presented in an article.35 The advantage of annotation projects is that they provide an environment where several resources are used on a regular basis over an extended period of three to six months. Thus, the students have the opportunity to investigate the range of facilities these resources offer. In addition, a biocurator needs to understand not only the topic being curated, but also the experimental approaches being taken and have the confidence to reject data that does not have sufficient statistical evidence to curate. Thus, the complexity of manual curation provides an opportunity for students to gain experience in applying a variety of bioinformatics resources to answer a research question while gaining a deeper understanding of a specific biological area of interest.10 These annotation projects, therefore, provide unique, in-depth training in multiple bioinformatics resources in a research context. This contrasts with short courses that tend to focus on one particular tool or database rather than how they can be utilized in concert to answer research questions.\n\nIt has been well documented that active teaching methods, which require students to use a more problem-based approach, are best used for transference of theoretical knowledge into practical utilisation.36–38 As biocuration projects provide a supportive active learning environment, it is unsurprising that this study has demonstrated that these projects enable students to consolidate their theoretical knowledge of bioinformatics resources and understand how these can be used in a research setting. However, the success of active teaching methods also heavily depends upon the quality of the supervision.39 In this survey, one of the qualitative themes identified was the excellent level of support and supervision for these projects (Table S9). The students are provided with prompt feedback on the annotations they suggest, and they have weekly one-to-one or small groups meetings with expert biocurators. As such, each of these projects requires a considerable investment of time by expert curators, time which is not always justified by the curation output of the students. Peer review of the annotations by the students themselves would provide a more efficient biocuration project model, and this has been achieved, by a group at Texas A&M, through the establishment of an annotation competition.12 As significant resources are needed to train new biocurators before they are able to work independently,29 biocuration projects, such as the ones provided at UCL, provide substantial initial training for students as biocurators, Indeed, three of our respondents are currently working as biocurators after completion of their projects.\n\nIn further agreement with Hosmani et al. (2019),11 our survey also confirmed that all participants (whether undergraduate or postgraduate) felt that their biocuration project provided them with the opportunity to increase their understanding of a variety of experimental methods in addition to increasing their appreciation of bioinformatics resources. In addition, for many of the students these projects provided an opportunity to increase their understanding of a biological domain that they intended to research in their future careers. This suggests that biocuration projects could be used to capture the knowledge a new student acquires from their own literature review at the start of their PhD project. Our survey compliments that of other student curation projects and confirms that early research scientists are able to contribute to manual curation efforts to improve existing resources.10,11 However, an important aspect of these student projects is the curation topic and the need for clear annotation rules or guidelines. Over the past 10 years at UCL we have identified that the students are more comfortable curating microRNAs, for which we have very strict guidelines, than the role of proteins within complex signalling pathways, for which the GO Consortium guidelines are less well defined.\n\nOne of the biggest challenges to biocuration is the time-consuming nature of reviewing, analysing and organising vast amounts of existing biological knowledge, which creates a bottleneck in genomics research when coupled with limited funding for biocurator roles and lack of trainers.10,29 Furthermore, the technological advances that have occurred, and are continuing to occur, in the post-genomic era are generating a considerable volume of new data. Consequently, there is an ongoing need to ensure accurate and efficient integration of this data into open-access databases, so that it can be fully exploited. However, there is also a need to integrate well established knowledge, across all areas of biology and biomedicine, into these databases.\n\nOne of the ways the biocuration community is trying to increase the volume of curated data is through community biocuration initiatives.8-11,29,40 Annotation projects have the potential to recruit early career researchers to community curation roles. This was confirmed by our survey, with all respondents stating they would be interested in participating in a community biocuration initiative. Primarily our respondents were keen for an opportunity to utilise newly gained skills and continue their contribution to the GO. However, several students were motivated to volunteer to participate in future curation projects because of the satisfaction of knowing that their annotations were being shared and would be of benefit to the wider scientific community. Unfortunately, there are only a very few examples of successful community biocuration efforts8,10,12,41,42 and, therefore, the opportunities for the wider scientific community to contribute to this need are limited. One of the rate limiting factors in community curation is checking the accuracy of the submitted annotations by expert biocurators. The CACAO competition model has addressed this issue by ensuring annotations are first peer reviewed by other competitors, which reduces the number of errors that need to be corrected before the data is incorporated into a public database.12 As well as benefiting the individual, through continued professional development, community biocuration would result in an enriched coverage of the literature benefiting the wider research community. Another potential source of community curators is retired scientists. These experts may be willing to curate their own articles as well as other seminal articles describing the role of key gene products in their area of expertise.\n\nIt is essential for any database to have both a high level of data coverage and accuracy, which requires continuous, high quality contributions, in order to facilitate discovery. Without integration of raw data and new knowledge into these resources, the discoverability and re-use of this data would be impaired.8 Another challenge for data integration is that many published articles cannot be curated due to a lack of sufficient specific data, which is detrimental to curation efficiency. These biocuration projects alter the students’ perspective of annotation and this could lead to their future research being written with biocurators in mind. Manual curation is a key approach by which databases are able to enhance and remain relevant to investigators and as such is a critical part of scientific work.43 Indeed, as the volume and breadth of data continues to increase, with advancements in both experimental methods and analysis techniques, curated data is fast becoming an essential resource in biomedical research.10,29\n\n\nConclusion\n\nThere is now accumulating evidence that student biocuration projects not only promote better understanding of molecular and cellular biology but can also contribute valuable high-quality annotations that improve existing resources.9-12 All of the respondents would recommend this project to future students, regardless of study stage or intended future career with respondent 3445942 stating: “I would highly recommend bioinformatics-based projects for students. It’s an area that is growing tremendously leading to many different career paths in life sciences. It is an important component that is found in any genetics data analysis field.” It is clear from our data that students gain substantial benefits by undertaking biocuration projects, ranging from improving their ability to carry out literature reviews and critically analyse articles, and extending their understanding of their chosen biological research area, to learning how to exploit bioinformatics resources. Furthermore, these projects lead to an increased appreciation and understanding of the essential and growing role of bioinformatics in scientific investigation. These biocuration projects, therefore, substantially contribute to the student’s overall education.\n\nAnnotation projects also benefit the wider biocuration community through a substantial increase in the number of annotations; a total of 5209 annotations have been added to 874 entities following students reviewing 529 articles. Additionally, the students provide a cohort with experience of biocurators who are willing and able to add to the GO knowledgebase throughout their careers through community biocuration initiatives. The importance of continued biocuration cannot be understated and as such, training of potential future bioinformaticians and curators should be a priority for any institution.\n\n\nData availability\n\nFigshare: Student biocuration projects as a learning environment.xlsx, https://doi.org/10.6084/m9.figshare.16629043.46\n\nThis project contains the following underlying data:\n\n- Table S1B: Raw data responses from participants to survey questions.\n\n- Table S2: Summary of participant demographics.\n\n- Table S3: Summary of responses to usefulness and enjoyablity of general (blue) and specific (green) project aspects.\n\n- Table S8: Student negative responses to biocuration project (Questions 7 and 8).\n\n- Table S9: Meaning Units assigned to each qualitative theme identified through text condensation methodology.\n\nFigshare: Student biocuration projects as a learning environment.xlsx, https://doi.org/10.6084/m9.figshare.16629043.46\n\nThis project contains the following extended data:\n\n- Table S1A: Questions included in the Opinio survey, with format of answer options and coding associated with specific answers.\n\n- Table S4: F-tests carried out for usefulness and enjoyability of general and specific aspect variance as well as post and prior bioinformatics knowledge with or without attendance of the GHD bioinformatics module.\n\n- Table S5: t-tests for comparison of usefulness and enjoyability of general and specific project aspects as well as post and prior bioinformatics knowledge with or without attendance of the GHD bioinformatics module.\n\n- Table S6: Chi2 tests for comparison of expected and observed responses to usefulness and enjoyabliity of project aspects as well as learning outcomes.\n\n- Table S7: ANOVA comparison of means for prior and post project bioinformatics knowledge and attendance of GHD bioinformatics module.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe are extremely grateful to all of the students who contributed GO annotation and completed the survey and to Alexander Deng for agreeing to the inclusion of his annotation histogram in this article. 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Publisher Full Text\n\nKirkpatrick JD, Kirkpatrick WK: Kirkpatrick’s four levels of training evaluation. Association for Talent Development. Alexandria, VA: ATD Press; 2016.\n\nMalterud K: Systematic text condensation: a strategy for qualitative analysis. Scand. J. Public Health. 2012; 40: 795–805. PubMed Abstract \| Publisher Full Text\n\nBalakrishnan R, Harris MA, Huntley R, et al.: A guide to best practices for Gene Ontology (GO) manual annotation. Database (Oxford). 2013; 2013: bat054. Publisher Full Text\n\nAndres H: The role of active teaching, academic self-efficacy, and learning behaviors in student performance. J. Int. Educ. Bus. 2020; 13: 221–238. Publisher Full Text\n\nBallen CJ, Wieman C, Salehi S, et al.: Enhancing Diversity in Undergraduate Science: Self-Efficacy Drives Performance Gains with Active Learning. CBE Life Sci. Educ. 2017; 16: ar56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreeman S, et al.: Prescribed active learning increases performance in introductory biology. CBE Life Sci. Educ. 2007; 6: 132–139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiggs JB, Tang CS: Teaching for quality learning at university: what the student does. McGraw-Hill, Society for Research into Higher Education & Open University Press; 2011.\n\nDedhia M, Kohetuk K, Crusio WE, et al.: Introducing high school students to the Gene Ontology classification system. F1000Res. 2019; 8: 241. Publisher Full Text\n\nVelankar S, Burley SK, Kurisu G, et al.: The Protein Data Bank Archive. Methods Mol. Biol. 2021; 2305: 3–21. Publisher Full Text\n\nNaithani S, et al.: Plant Reactome: a knowledgebase and resource for comparative pathway analysis. Nucleic Acids Res. 2020; 48: D1093–D1103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOdell SG, Lazo GR, Woodhouse MR, et al.: The art of curation at a biological database: Principles and application. Curr. Plant Bio. 2017; 11-12: 2–11. Publisher Full Text\n\nBinns D, et al.: QuickGO: a web-based tool for Gene Ontology searching. Bioinformatics. 2009; 25: 3045–3046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLong JM, Ray B, Lahiri DK: MicroRNA-153 physiologically inhibits expression of amyloid-β precursor protein in cultured human fetal brain cells and is dysregulated in a subset of Alzheimer disease patients. J. Biol. Chem. 2012; 287: 31298–31310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLovering RC, Thurlow KE, De Miranda Pinheiro S: Student biocuration projects as a learning environment.xlsx. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "96513", "date": "27 Oct 2021", "name": "Nicole A. Vasilevsky", "expertise": [ "Reviewer Expertise Biocuration", "biomedical ontology development", "ontology", "and data management training" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nicely describes student perceptions of a biocuration training course at the University College of London.\nThis paper is well written and provides nice examples of the benefits of this course, through the increased GO annotations contributed by the students. The survey results provide evidence for the usefulness and the positive impact of the course.\nIn the introduction, it would be great to have a more detailed description of your definition of bioinformatics and its relationship to the biocuration field.\nIt would be nice to read about how the students performed in the course. Were they all successful at performing quality annotations or did the quality vary amongst the students? Did it improve over time? If you were to perform a future study on the outcomes of this training, it would be nice to see inter-curator agreement between students and between the student and expert curator/mentor.\nIt would be interesting to hear more about the community biocuration effort that was offered as a follow-up to this course.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7356", "date": "28 Oct 2021", "name": "Ruth Lovering", "role": "Author Response", "response": "We thank the reviewer for these positive comments and interest in our article. A definition of bioinformatics is challenging, the course we provide the students only touches on this field, the areas covered are all mentioned in the survey. As biocuration provides biological annotations it is certainly is an essential component of many aspects of bioinformatics. The majority of students were able to provide very good annotations for at least 15, but often 30 articles. However, there was considerable variability in how much feedback was required during the project and how quickly the student reached an understanding that enabled them to create high quality annotations. For some students this took around a month, for others it was only towards the end of the project that this was achieved. Inter-curator annotation consistency is an interesting discussion point, especially as it can be difficult to achieve this in annotation groups. The number of students undertaking annotation projects at UCL each year prevents this from an area we can investigate; the students are required to submit a dissertation and do not have time to review another student’s annotations. However, Ramsey et al. (in press) discuss a long running project which involves students assessing other students’ annotations. Only a few students have continued to provide annotations after the end of their annotation project and consequently there is very little to report on this." } ] }, { "id": "99639", "date": "02 Dec 2021", "name": "Anna Delprato", "expertise": [ "Reviewer Expertise Systems Genetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Thurlow et al. describes the utility of implementing a biocuration project to teach students how to critically evaluate primary scientific literature, and at the same time contribute annotations to the Gene Ontology database, which is a valuable resource used by the scientific community worldwide. The authors have provided data evaluating student experience and project enjoyability, as well as learning outcomes. This is an interesting and useful project design, which is clearly beneficial to students and the research community. I have several comments that need to be addressed.\nIntroduction:\n\"utilised in a plethora of future careers\" - Please indicate which careers these skills could be useful for.\n\n\"Recent technological advancements in high-throughput research have resulted in vast amounts of data output, which is outstripping analysis capabilities\" - Please indicate which technological advances specifically?\n\nMethods:\nThe authors should explain how the literature searches for annotation are performed.\n\nHow are the articles selected?\n\nAre students involved in article selection?\n\nIf not, do they learn this as part of the training?\n\nAre Pubmed, Google Scholar, and MeSH used to find articles for curation?\n\nWhat is the criteria for article selection?\n\nAre the articles mostly current?\n\nAre the use of certain methods a factor in the selection process?\n\nOther items:\nReference 31 the citation link is broken\n\nSupplementary Workbook:\nTable S2. \"undertaken\" is misspelled in the title.\n\nAuthors should recheck spelling throughout the supplementary workbook.\n.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "7618", "date": "02 Feb 2022", "name": "Ruth Lovering", "role": "Author Response", "response": "We appreciate the suggestions made by Dr Delprato. The revisions we have made to address Dr Delprato concerns are listed below. We hope that these are sufficient to address these points. Introduction: 1a) \"utilised in a plethora of future careers\" - Please indicate which careers these skills could be useful for. Manuscript revisions In the first paragraph of the introduction, we have provided a short list of potential careers: ‘As such, a research project in bioinformatics requires the incorporation of skills from a multitude of areas and can be utilised in a plethora of future careers, such as computational biologists, bench scientists, project managers, copywriters, clinicians, bioinformaticians or administrators.’ In addition, we have added a list of transferable skills that students will have developed after completing a biocuration project, at the start of the last paragraph of the introduction: ‘Students develop a range of transferable skills during the GO biocuration projects, that they would have developed when undertaking a bench or literature review project. For example, the students improve their writing and communication skills, their project and time management skills, as well as their ability to find and understand relevant data, critically evaluate this data and summarise the information.’ Methods: The authors should explain how the literature searches for annotation are performed. How are the articles selected? Are students involved in article selection? If not, do they learn this as part of the training? Are Pubmed, Google Scholar, and MeSH used to find articles for curation? What is the criteria for article selection? Are the articles mostly current? Are the use of certain methods a factor in the selection process? Manuscript revisions The method section has been revised and an additional supplementary table created (S10) has been created and the new version of the supplementary tables is available 10.6084/m9.figshare.16629043. This table lists the activities the student and supervisors undertake during the ‘curation’ part of the project. In the Methods, Participants and Setting section the following two paragraphs have been added: ‘All of the participants had undertaken a GO biocuration project, however, while the format for each project was generally the same, there was some variation. These variations were due to the study level of the student (which ranged from undergraduate to post-graduate), the interest of the student in learning key aspects of biocuration, the biological knowledge of the student, the title of the project, the project length (ranging from 4-8 months, full or part-time) and finally the timing of the project with respect to the Covid-19 pandemic. A list of the activities undertaken during the project are provided in Supplementary Table S10. All students chose a specific area of biology, usually linked to a specific disease, to curate. Some of the students focused on curating proteins, while others focused on microRNAs, but all students follow existing GO consortium guidelines26,35,47. For protein focused annotation projects, articles for curation are identified by a PubMed search using HUGO gene nomenclature (HGNC) approved symbols48, and key words (if there are more than 10 articles to review). Articles are only curated if there is sufficient information about the species of the protein used in the experiments and the article provides experimental data that describes the role of the protein in the disease or process that the student is curating. For microRNA projects the HGNC symbol of the microRNA target is used to search either miRTarBase49or emiRIT50. If no suitable articles are identified, then PubMed is searched with the HGNC symbol and key words such as microRNA (Supplementary Table S10). Articles are only curated if there is evidence that the microRNA binds to the target mRNA that encodes for one of the prioritised proteins (usually this is provided by a reporter assay) and there is sufficient information about the species of the microRNA and mRNA used in the experiments. The publication date is not usually used to filter articles to curate.’ Other items: Reference 31 the citation link is broken Manuscript revision I am unable to edit the citation links. Also the new references added are not linked to the reference list and are also out of sequence. Supplementary Workbook: Table S2. \"undertaken\" is misspelled in the title. Authors should recheck spelling throughout the supplementary workbook. Manuscript revision The supplementary tables have been checked and all spelling mistakes corrected. The new version of the supplementary tables is available 10.6084/m9.figshare.16629043." } ] } ]	1	https://f1000research.com/articles/10-1023
https://f1000research.com/articles/11-128/v1	01 Feb 22	{ "type": "Research Article", "title": "Effects of asparaginases and L-carnitine on Western-diet-induced hepatosteatosis in mice", "authors": [ "Mona Ali Mahmoud Assar", "Martina Hüffel", "Mamdouh Afify", "Ralf Weiskirchen", "Albrecht Eisert", "Rene Tolba", "Julia Steitz", "Mona Ali Mahmoud Assar", "Martina Hüffel", "Mamdouh Afify", "Ralf Weiskirchen", "Albrecht Eisert", "Rene Tolba" ], "abstract": "Abstract Background: Asparaginases are common chemotherapeutic agents used for the treatment of acute lymphoblastic leukemia as a single or combinational therapy. Accompanying hepatotoxicity makes its use in elderly patients with pre-conditions, as obesity or other hepatopathies, difficult. Various hepatoprotective compounds like, L-carnitine, are discussed to ameliorate the induced hepatotoxicity. Methods: Here we aimed to establish a mouse model to study the effect of asparaginases (L-asparaginase and Oncaspar) and L-carnitine on Western-diet-induced hepatosteatosis in mice. Dose-escalation studies were performed to analyze asparaginases induced hepatotoxicity in C57BL/6 mice with normal or fatty livers. Subsequently, the effect of L-carnitine to improve the induced toxicity was tested. Results: Our results showed mild-to-moderate hepatotoxic effects while the Western-diet induced a higher degree of vacuolization and hepatocyte damage in liver tissue. Testing of L-carnitine in the established models did not show any protective effect on the toxicity or impairment of the efficacy of asparaginases. Conclusion: The here established models were able to demonstrate the asparaginase-induced hepatotoxic effects which were enhanced by the Western-diet. However, to test potential ameliorating drugs, the models might need some improvements.", "keywords": [ "asparaginases", "hepatosteatosis", "L-carnitine", "hepatotoxicity." ], "content": "Introduction\n\nThe bacterial-derived enzyme asparaginase has been successfully used in chemotherapeutic regimens developed for the treatment of acute lymphocytic leukemia (ALL) in children and adult patients.1,2 Clinically, three common forms of asparaginase are in use: a native L-asparaginase isolated from Escherichia coli, a pegylated form (polyethylene glycol asparaginase, also called pegaspargase or Oncaspar) and an Erwinia chrysanthemi derived asparaginase known as Erwinia asparaginase (Erwinase).2 Pegaspargase has been developed to reduce the immunogenicity of the enzyme and prolong its half-life in plasma.3\n\nDespite the essential role of L-asparaginase as a chemotherapeutic agent, adverse effects have been reported and comprise hypersensitivity reactions, liver injury, pancreatic toxicity, coagulopathy, immune suppression and effects on the central nervous system resulting in the decrease of L-asparagine and L-glutamine levels.4\n\nEven though asparaginases are efficiently applied as combinational chemotherapeutic agents in children’s and adults’ ALL, these drugs can induce side effects mainly in elderly patients with pre-existing conditions such as hepatopathies.5 With aging, people often become overweight and obese increasing the risk of additional health problems and co-morbidities.5 Previous studies have reported that patients with preceding hepatic steatosis or hepatitis, developed more severe side effects after treatment with chemotherapeutic drugs6,7 or showed even fatal liver failure.8 Various compounds e.g., L-carnitine (L-C) have been described to ameliorate liver damage in patients with non-alcoholic fatty liver disease.9–11 Additionally, L-C reduced kidney and heart toxicity triggered by cisplatin and doxorubicin chemotherapeutic drugs.12,13\n\nWe aimed in our experiments to establish a fatty liver model in mice to study the effects of asparaginases on the liver and the potential intervention with L-C to ameliorate toxicity.\n\n\nMethods\n\nAnimal studies were approved by the Governmental Animal Care and Use Committee at the LANUV NRW, Recklinghausen, Germany (approval number: AZ 87-51.04.2010.A278 and AZ 84-02.04.2016.A433), and performed in accordance with the German legislation governing animal studies following the guide for the care and use of Laboratory Animals (NIH publication, 8th edition, 2011) and the Directive 2010/63/EU on the protection of animals used for scientific purposes. Our animal study was conducted according to the ARRIVE guidelines 2.0 using the ARRIVE Essential 10 checklist for pre-clinical animal studies.\n\n6-week-old female and, 3–5 weeks old male C57BL/6NCrl mice were obtained from Charles River, Sulzfeld, Germany. Our best efforts were made to minimize suffering and pain of animals by applying 3Rs principle in our animal experiments. Water and diet were supplied ad libitum and animals were housed under specific-pathogen-free condition according to the Federation of European Laboratory Animal Science Associations (FELASA) guidelines. Mice were housed in a barrier housing facility at the Institute for Laboratory Animal Science in Aachen, Germany. The room temperature was 21–23 °C with a humidity of 30 –70 %. The light intensity was below 200 lux at a height of 1 m and a light-dark rhythm of 12 hours each. Animals were inspected on daily basis. The animals were kept (max. 5 mice/cage) in type II L cages according to the requirements of Annex 3 of the EU Directive 2010/63. A total of 40 female mice were utilized for dose-escalation study with Oncaspar® (Sigma-Tau, Rome, Italy) and fed for the first 4 weeks with either normal (normal liver, NL) or Western-diet (fatty liver, FL) (Sniff Spezialdiäten GmbH, Soest, Germany). After feeding duration, animals were randomly divided into eight groups (n=5). Five animals per group received 5 intravenous injections of Oncaspar® with either 2700 U/kg, 4050 U/kg, and 5400 U/kg bodyweight into the lateral tail vein in a maximum volume of 5 mL/kg body weight every two days. Control groups received corresponding volumes of 0.9% NaCl solution (Figure 1A).\n\n* p < 0.05, p < 0.01, * p < 0.001, **** p< 0.0001.\n\nAnalysis of L-asparaginase (Spectrila®, Medac, Wedel, Germany) was performed in male mice after 8 weeks of Western-diet or normal diet. A total number of 120 animals were randomly divided into 6 groups (n=20). Twenty animals/group received 5 doses of 5400 U/kg bodyweight L-asparaginase or 0.9% NaCl solution as control every two days (Figure 3A). In indicated groups, animals received L-C (300 mg/kg bodyweight) intraperitoneally.\n\nBodyweight and health conditions were measured daily during the treatment period up to day 4 or day 28 after the last treatment. A modified version of the scoring system described by Morton and Griffiths14 was utilized twice a week during feeding duration and daily during injection duration for assessment of severity scoring. Animals were anesthetized for final procedure with inhalation anaesthesia using isoflurane. The maximum amount of blood was collected from retro-orbital sinus under isoflurane anaesthesia using capillary tubes and finally sacrificed with an overdose of isoflurane and organs were collected for further analysis.\n\nBodyweight changes were calculated based on the bodyweight of the animal at day 0 (before treatment start) and the necropsy time points (days 4 and 28) and were expressed in percentage.\n\nWhite blood cell counts (WBCs) were measured in K-EDTA blood received from the final retro-bulbar blood draw using the Celltac α MEK-6550K (Nihon Kohden Europe GmbH, Rosbach vor der Höhe, Germany) analyzer. Indicated serum parameters were analyzed in serum using the VITROS 350 (Ortho Clinical Diagnostics, Neckargemünd, Germany) system.\n\nHematoxylin and eosin staining\n\nLiver samples were fixed in 4% neutral buffered formaldehyde (Carl Roth GmbH, Karlsruhe, Germany) for 48 h, dehydrated and embedded in paraffin. 4–6 μm thick liver sections were stained with hematoxylin and eosin (H&E). Grading of liver damage was performed by a board-certified pathologist in a blinded manner in five randomly selected fields in each liver specimen at 400× magnification. A scale of 1–5 was utilized for grading of lesions; 1=no or negligible changes, lesions affecting 0–10% of the field; 2=mild, lesions affecting 10–30% of the field; 3=moderate, lesions affecting 30–60% of the field; 4=moderate to severe, lesions affecting 60–80% of the field; and 5=severe, lesions affecting >80% of the field. Different histopathological changes were examined: hepatocytic vacuolization, degenerative changes, congestion, cellular infiltration, and Kupffer cell activation.\n\nOil Red O stain\n\nStandard Oil Red O (ORO) stain (Merck, Darmstadt, Germany) was used for the detection of neutral triglycerides and lipids in 5 μm cryo-sections of the liver embedded in Tissue-Tek (Sakura Finetek, Torrance, CA, USA). ImageJ software (ImageJ, RRID:SCR_003070) was utilized for quantification of accumulated lipids. Five bright-field images were captured per slide using a light microscope at 200× magnification.\n\nStatistical analysis was performed using GraphPad Prism 8 software (RRID:SCR_002798) (GraphPad Software Inc., San Diego, CA, USA) and results are expressed as Mean ±SD. One-way analysis of variance (ANOVA) for multiple comparisons between the mean of different treatment groups and Bonferroni correction test was used. Results were considered significant for p-values <0.05.\n\n\nResults\n\nFirst, a dose-escalation study with Oncaspar was performed to evaluate the hepatotoxic effect after five intravenous injections in normal (normal liver) or Western-diet (fatty liver) fed female mice (Figure 1A).\n\nWestern-diet feeding for 4 weeks resulted in a not significant elevation of bodyweight changes compared to the control group fed with a normal diet. All doses of Oncaspar induced a marked reduction in bodyweight changes in all groups compared to their controls (Figure 1B). To evaluate the efficiency of the chemotherapeutic drug Oncaspar, WBC were measured showing significant reduction in all Oncaspar treatment groups (except for FL+ Oncaspar 4050) compared to their control groups (Figure 1C).\n\nFor the evaluation of toxicity, clinical chemistry parameters in serum were analyzed and are shown in Figure 1 and Table 1. Significant reduction in serum albumin (ALB), as a marker of hepatic protein synthesis, is observed in all Oncaspar treatment groups (Figure 1D) and similar results were observed for total protein (TP) (Table 1). Parameters for liver toxicity showed no significant elevations in aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels due to the Western-diet. Oncaspar itself did not influence the levels of AST and LDH compared to their control groups (Figure 1E and F). However, Oncaspar induced a significant elevation in serum amylase, a parameter indicating also pancreatic toxicity, in the FL treatment groups (Figure 1G). The efficient induction of a fatty liver in mice (FL group) was verified also based on the significant increase of serum cholesterol (Figure 1H). On the other hand, Oncaspar treatment induced a non-significant decrease in cholesterol in both NL and FL animal groups when compared to their non-treated controls.\n\nFor characterization of the degree of liver damage induced by various doses of Oncaspar as well as Western-diet, histopathological analyses were performed via scoring of the pathologic alteration in liver tissue samples. Here, higher levels of degenerative changes in hepatocytes as well as hepatocytic vacuolization were observed in livers after Oncaspar administration (Figure 2) and were even more pronounced and significant in the fatty liver groups (Figure 2C).\n\nArrows refer to hepatocytic vacuolization (A). Evaluation of hepatocytic vacuolization (B), degenerative changes (C) and Oil Red O (ORO) staining (D) of liver sections are shown as means± SD. Arbitrary units (a.u.). * p < 0.05, p < 0.01, * p < 0.001, **** p< 0.0001.\n\nIn addition, the semi-quantitative evaluation of ORO stained cryo-sections visualizing the amount of accumulated lipids in hepatocytes which showed significant accumulation of lipid droplets in hepatocytes cytoplasm in all FL animal groups, while only little lipid accumulation was observed in livers of the NL groups (Figure 2D).\n\nAlthough the dose-escalation study with Oncaspar in female C57BL/6 mice showed already signs of hepatotoxicity in normal and Western-diet fed mice, we decided to establish a second more stringent model to induce a higher degree of hepatotoxicity to better discriminate between the fatty liver and normal liver group and the chemotherapeutic treated and control groups. Based on prior described observation and published data, we choose to increase the feeding time (8 instead of 4 weeks) and to use male instead of female mice. In addition, we tested L-asparaginase, a compound commonly used for the treatment of ALL and known to induce liver toxicity accompanied by steatosis, especially in elderly patients. In order to ameliorate the hepatotoxic effect of L-asparaginase, groups of L-carnitine treatment were added (Figure 3A). As shown in Figure 3B, bodyweight changes at day 4 after the last treatment indicated that L-C and L-asparaginase treatment resulted in a bodyweight loss which was significantly higher after L-asparaginase administration in FL groups. Interestingly, L-C was not able to ameliorate the effect of L-asparaginase in reducing bodyweight in FL animal groups. Efficiency of L-asparaginase to reduce WBCs is demonstrated in Figure 3C showing no impact of the L-C treatment.\n\n* p < 0.05, p < 0.01, * p < 0.001, **** p< 0.0001.\n\nUnlike Oncaspar, the same dosage of L-asparaginase did not decrease ALB levels in NL and FL animal groups at the 4-day time point. Even a significant increase in ALB levels is observed in animals with a fatty liver and L-asparaginase+L-C treatment (Figure 3D). AST measurements exhibited a slight elevation in FL animal group, which was significant in the L-asparaginase+L-C group compared to the NL control groups (Figure 3E). LDH enzyme levels were not significantly different between the L-asparaginase or L-C treatment groups. However, a consistent increase is observed due to the Western-diet (Figure 3F). The amylase measurement showed a highly significant increase in the FL groups independent of the L-asparaginase or L-C treatment (Figure 3G). Similar results were obtained for cholesterol levels (Figure 3H). Further analysis of clinical chemistry parameters at the 4-day time point are given in Table 2.\n\nFor the detection of possible late sequestration and/or reversible effects, further animals were analyzed on day 28 after the last application of L-asparaginase. Here, WBCs levels went back to normal and no further changes or reversible effects in serum parameters are found (Table 3).\n\nHistopathological analysis revealed a significant increase in hepatocytic vacuolization and in degenerative changes in all FL groups (Figure 4B/C) at the 4-day time point. Furthermore, FL animals displayed more severe degenerative changes in hepatocytes after L-asparaginase and L-C (FL L-asparaginase+L-C) treatment compared to normal livers from the NL L-asparaginase+L-C treatment group (Figure 4C), indicating that Western-diet deteriorates the effect of L-asparaginase on hepatocytes. In addition, focal areas of necrosis were observed in liver tissue sections of L-asparaginase treated animals independent of the L-C treatment in all groups. Lipid accumulation evaluated in the ORO staining showed significant increase in all FL animal groups and was further increased by the L-asparaginase treatment (Figure 5).\n\nArrows refer to focal area of necrosis. Arrow heads indicate post necrotic area (A). Hepatocytic vacuolization (B), degenerative changes (C) were evaluated and depicted as means±SD. * p < 0.05, p < 0.01, * p < 0.001, **** p< 0.0001.\n\n* p < 0.05, p < 0.01, * p < 0.001, **** p< 0.0001.\n\nLonger feeding with Western-diet resulted in main aggregation of vacuolization in midzonal and centrilobular areas of the liver as depicted in tissue sections at the 28-day time point (Figure 6). Again, differences in hepatocytic vacuolization, degenerative changes and lipid accumulation were even more prominent between livers of the NL and FL groups and worsened after L-asparaginase and L-C treatment (Figure 6B-D).\n\nArrows refer to focal area of necrosis (A). Hepatocytic vacuolization (B) and degenerative changes (C) were evaluated and shown as means±SD. * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001.\n\n\nDiscussion\n\nEven though asparaginases are efficiently used as chemotherapeutic drugs side effects especially in elderly patients with pre-existing conditions can counteract their unrestricted use.5 It has been shown that non-healthy livers are more susceptible to chemotherapy-induced adverse effects15,16 and that chemotherapeutic drugs cause liver toxicity accompanied by steatosis especially in older patients.5 Therefore, we aimed to develop a fatty liver (steatotic) model mimicking the situation in humans to study the effect of asparaginases induced liver toxicity in C57BL/6 mice to test L-carnitine as a protective agent to ameliorate asparaginase-induced liver toxicity.\n\nBodyweight changes, blood parameters and clinical chemistry as well as histopathological changes in the livers of mice were analyzed and efficacy of the chemotherapeutic drug was verified by counting the WBCs. Asparaginases were reported to cause reduction in bodyweight17 due to increased catabolism,18 which could be also verified in our models in all treatment groups. As expected, the WBCs count showed a marked reduction after administration of Oncaspar or L-asparaginase in all treatment groups.19\n\nClinical chemistry parameter measurement showed a significant decline in albumin and total protein levels after Oncaspar administration, independent of the condition of the liver by inhibition of protein synthesis associated with the main mechanism of the drug.20 Hypoalbuminemia is one of the major abnormalities associated with Oncaspar treatment21 with a consequent hepatic toxicity. In contrary, treatment of L-asparaginase resulted in a significant elevation of albumin in FL L-asparaginase+L-C group. This might be attributed to the fact that L-asparaginase is known to have a shorter half-life than Oncaspar22 that consequently enhances clearance rate in the circulatory system via native proteases.23\n\nALT and AST levels in serum indicating liver toxicity, showed only non-significant slight elevations, mainly due to the Western-diet. Our results were in line with previously published data showing no signs of hepatotoxicity after treating mice with Oncaspar.17\n\nAmylase, as a marker of pancreatic toxicity, showed a significant increase in FL animals treated with Oncaspar especially in the highest dosage group. However, the model using L-asparaginase showed only amylase elevation due to the Western-diet suggesting that feeding animals with high fat diet stimulate the secretion of amylase as previously described.24 Several investigations argued whether asparaginases attributed to hypercholesterolemia25 or hypocholesterolemia.26 Our models demonstrated a significant increase of cholesterol in animals fed with Western-diet and has been expected and reported in another study.27 The administration of Oncaspar seemed to reduce this increase, while L-asparaginase did not show any effect of the Western-diet-induced cholesterol elevation.\n\nHistopathological analysis of H&E-stained liver tissue sections demonstrated hepatocytic degenerative changes, like vacuolization, as a main finding of hepatotoxicity associated with Oncaspar administration but also with Western-diet alone (Figure 2). Hepatic changes reached to a moderate severity grade and degenerative changes were observed in a dose-dependent manner after Oncaspar treatment in NL and FL groups showing a deteriorating effect in the FL groups. These results confirm former investigations in an ex vivo model, in which a higher liver toxicity in histopathological analysis induced by L-asparaginase treatment in fatty liver was reported.28\n\nThe second model using L-asparaginase histopathological analysis revealed that the observed significant increase of hepatic vacuolization was mainly due to the Western-diet. In regard to the degenerative changes, animals of the NL groups treated with L-asparaginase showed also mild changes and scored higher in the FL groups.\n\nORO-stained liver sections showed a significant accumulation of lipids due to the Western-diet and the administration of Oncaspar, at least in the highest dosage group. Thus, Oncaspar treatment deteriorated the effect of Western-diet in accumulating lipids and our model showed a clear sign of hepatosteatosis. L-asparaginase treatment of Western-diet fed animals showed hereby similar results (Figure 5). As already described asparaginases induced hepatotoxicity is attributed to inhibition of protein synthesis and related to mitochondrial dysfunction8 and impairment of energy production leading to micro-vesicular steatosis, hepatocytes necrosis or apoptosis.29 Lipoproteins and lipids get exported and accumulate in hepatocytes resulting in steatosis and liver dysfunction.30\n\nVacuolization mainly aggregated in midzonal and centrilobular areas of liver tissues. We noticed that more severe vacuolization of hepatocytes depicted at the 28-day time point in FL animal groups was a result of longer feeding with Western-diet. We could demonstrate the impact of Oncaspar and/or L-asparaginase treatment in worsening the effect of Western-diet in accumulating lipids in hepatocytes and thus our model showed a clear sign of hepatosteatosis. Our findings were in compliance with former studies in which accumulation of lipids in hepatocytes after feeding mice with high fat content diet was observed resulting in hepatic steatosis.31\n\nTo improve the animal model in order to discriminate better between the effects of Western-diet and chemotherapy, the feeding time was increased from 4 weeks to 8 weeks and male mice, which are described as more vulnerable for the induction of fatty liver,32 were used. Male mice develop rather symptoms of human metabolic syndrome than females,33 due to estrogen which provide a protective capacity against non-alcoholic fatty liver disease.34 For these reasons, we substituted females with males in our experiment to develop an animal model showing metabolic syndrome mimicking those of overweight humans.\n\nDue to the antioxidant properties of L-carnitine it has been utilized as a protective agent against toxicity that might be triggered by cisplatin or doxorubicin as anti-cancer drugs.35 In our model, L-C was not able to ameliorate the effect of L-asparaginase on bodyweight changes in FL animal groups. As already described it also had no effect on the efficacy of L-asparaginase in reducing WBC’s in our model.17 However, L-C administration resulted in a non-significant increase in our tested clinical chemistry parameters. Histopathological findings did not show significant improvement based on the evaluation of hepatocytic vacuolization. On the contrary, degenerative changes was even deteriorated after L-C application in FL animal groups after asparaginase treatment. Our findings were in line with previous studies that proved ineffectiveness of L-C in ameliorating parameters referring to hepatic dysfunction as well as impairing hepatic steatosis histologically in a C57BL/6 mouse model.36\n\nIn summary, our established models showed mild toxic effects of Oncaspar with slight to moderate liver toxicity. In Western-diet-fed animals, a higher degree of hepatocyte damage and vacuolization could be observed in liver tissue compared standard diet fed animals. Only mild signs of liver toxicity could be observed after using L-asparaginase, while more (but not significant) severe signs, were detected in animals with fatty livers. L-C could not ameliorate the induced toxicity and did not show an impairment of the efficacy of Oncaspar or L-asparaginase. However, with the mild severity grade of the liver toxicity model it might be not severe enough to discriminate between effects induced by the Western-diet and chemotherapeutic drug in order to subsequently test potential ameliorating drugs.\n\n\nData availability\n\nFigshare: Underlying data for ‘Effects of asparaginases and L-carnitine on Western-diet-induced hepatosteatosis in mice’\n\nMeasured parameters of dose-escalation study of Oncaspar. https://doi.org/10.6084/m9.figshare.17032946.37\n\nMeasured parameters of L-asparaginase study at the day 4 time point. https://doi.org/10.6084/m9.figshare.17032391.38\n\nMeasured parameters of L-asparaginase at the 28 days’ time point. https://doi.org/10.6084/m9.figshare.17068124.39\n\nResults of histopathological analysis. https://doi.org/10.6084/m9.figshare.17068148.40\n\nFigshare: ARRIVE guideline checklist for ‘Effects of asparaginases and L-carnitine on Western-diet-induced hepatosteatosis in mice https://doi.org/10.6084/m9.figshare.18620330.41\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\nAvramis VI, Tiwari PN: Asparaginase (native ASNase or pegylasted ASNase) in the treatment of acute lymphoblastic leukemia. Int. J. Nanomedicine. 2006; 1(3): 241–254. PubMed Abstract\n\nPieters R, et al.: L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase.2011; 117(2): p. 238–249.\n\nAvramis VI, et al.: A randomized comparison of native Escherichia coli asparaginase and polyethylene glycol conjugated asparaginase for treatment of children with newly diagnosed standard-risk acute lymphoblastic leukemia: a Children's Cancer Group study. Blood. 2002; 99(6): 1986–1994. PubMed Abstract \| Publisher Full Text\n\nChabner BA, Loo TJCCP; Practice: Enzyme therapy: L-asparaginase.1990; p. 397–407.\n\nEarl M: Incidence and management of asparaginase-associated adverse events in patients with acute lymphoblastic leukemia. Clin. Adv. Hematol. Oncol. 2009; 7(9): 600–606. PubMed Abstract\n\nMori T, et al.: Histopathologic features of the biopsied liver at onset of childhood B-precursor acute lymphoblastic leukemia presenting as severe jaundice.1997; 25(3): p. 354–357.\n\nSoh L-T, et al.: Fulminant hepatic failure in non-Hodgkin lymphoma patients treated with chemotherapy.1992; 28(8-9): p. 1338–1339.\n\nBodmer M, et al.: Fatal liver failure in an adult patient with acute lymphoblastic leukemia following treatment with L-asparaginase. Digestion. 2006; 74(1): 28–32. PubMed Abstract \| Publisher Full Text\n\nChang B, et al.: L-carnitine inhibits hepatocarcinogenesis via protection of mitochondria.2005; 113(5): p. 719–729.\n\nMalaguarnera M, et al.: L-carnitine supplementation to diet: a new tool in treatment of nonalcoholic steatohepatitis—a randomized and controlled clinical trial.2010; 105(6): p. 1338–1345.\n\nSocha P, et al.: Pharmacological interventions for nonalcoholic fatty liver disease in adults and in children: a systematic review.2009; 48(5): p. 587–596.\n\nAlshabanah OA, et al.: Doxorubicin toxicity can be ameliorated during antioxidant L-carnitine supplementation.2010; 3.\n\nTufekci O, et al.: Evaluation of the effect of acetyl L-carnitine on experimental cisplatin nephrotoxicity.2009; 55(6): p. 451–459.\n\nMorton DB, Griffiths PH: Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Vet. Rec. 1985; 116(16): 431–436. Publisher Full Text\n\nHardwick RN, et al.: Increased susceptibility to methotrexate-induced toxicity in nonalcoholic steatohepatitis. Toxicol. Sci. 2014; 142(1): 45–55. PubMed Abstract \| Publisher Full Text\n\nSchroder T, et al.: Liver steatosis is a risk factor for hepatotoxicity in patients with inflammatory bowel disease under immunosuppressive treatment. Eur. J. Gastroenterol. Hepatol. 2015; 27(6): 698–704. PubMed Abstract \| Publisher Full Text\n\nSea JL, et al.: Levocarnitine does not impair chemotherapy cytotoxicity against acute lymphoblastic leukemia.2020; 61(2): p. 420–428.\n\nNikonorova IA, et al.: Obesity challenges the hepatoprotective function of the integrated stress response to asparaginase exposure in mice. J. Biol. Chem. 2017; 292(16): 6786–6798. PubMed Abstract \| Publisher Full Text\n\nBunpo P, et al.: Alanyl-glutamine consumption modifies the suppressive effect of L-asparaginase on lymphocyte populations in mice. J. Nutr. 2008; 138(2): 338–343. PubMed Abstract \| Publisher Full Text\n\nSahoo S, Hart J: Histopathological features of L-asparaginase-induced liver disease. Semin. Liver Dis. 2003; 23(3): 295–299. PubMed Abstract \| Publisher Full Text\n\nLiu C, et al.: Asparaginase potentiates glucocorticoid-induced osteonecrosis in a mouse model.2016; 11(3): p. e0151433.\n\nAsselin BL, et al.: Comparative pharmacokinetic studies of three asparaginase preparations. J. Clin. Oncol. 1993; 11(9): 1780–1786. PubMed Abstract \| Publisher Full Text\n\nNunes JC, et al.: Recent Strategies and Applications for l-Asparaginase Confinement.2020; 25(24): p. 5827.\n\nSaravanan G, et al.: Anti-obesity action of gingerol: effect on lipid profile, insulin, leptin, amylase and lipase in male obese rats induced by a high-fat diet.2014; 94(14): p. 2972–2977.\n\nKonya H, et al.: L-asparaginase-induced hyperlipidemia in a case of acute lymphoblastic leukemia. Rinsho Ketsueki. 1991; 32(3): 250–254. PubMed Abstract\n\nKose D, et al.: Effects of Prednisolone, L-Asparaginase, Gemfibrozil, and Combinations of These Elements on Mice Lipid Profile, Liver, and Pancreas. J. Pediatr. Hematol. Oncol. 2016; 38(1): e42–e49. Publisher Full Text\n\nGlastras SJ, et al.: Mouse Models of Diabetes, Obesity and Related Kidney Disease. PLoS One. 2016; 11(8): e0162131. PubMed Abstract \| Publisher Full Text\n\nRoesmann A, et al.: L-carnitine ameliorates L-asparaginase-induced acute liver toxicity in steatotic rat livers.2013; 59(3): p. 167–175.\n\nLabbe G, et al.: Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.2008; 22(4): p. 335–353.\n\nKamal N, et al.: Asparaginase-induced hepatotoxicity: rapid development of cholestasis and hepatic steatosis.2019; 13(5): p. 641–648.\n\nFeng D, et al.: Curcumin prevents high-fat diet-induced hepatic steatosis in ApoE−/− mice by improving intestinal barrier function and reducing endotoxin and liver TLR4/NF-κB inflammation. Nutr. Metab. 2019; 16(1): 79. PubMed Abstract \| Publisher Full Text\n\nIngvorsen C, et al.: The role of sex and body weight on the metabolic effects of high-fat diet in C57BL/6N mice.2017; 7(4): p. e261–e261.\n\nGallou-Kabani C, et al.: C57BL/6J and A/J mice fed a high-fat diet delineate components of metabolic syndrome.2007; 15(8): p. 1996–2005.\n\nIbrahim SH, et al.: Animal models of nonalcoholic steatohepatitis: eat, delete, and inflame.2016; 61(5): p. 1325–1336.\n\nBlanca AJ, et al.: Inflammatory and fibrotic processes are involved in the cardiotoxic effect of sunitinib: Protective role of L-carnitine. Toxicol. Lett. 2016; 241: 9–18. PubMed Abstract \| Publisher Full Text\n\nLiu Y, et al.: L-carnitine does not ameliorate asparaginase-associated hepatotoxicity in a C57BL6 mouse model. Leuk. Lymphoma. 2019; 60(8): 2088–2090. PubMed Abstract \| Publisher Full Text\n\nMona Ali Mahmoud A, et al.: Measured parameters of dose escalation of Oncaspar.2021.\n\nMona Ali Mahmoud A, et al.: Measured parameters in L-asparaginase experiment at 4 days time point.2021.\n\nMona Ali Mahmoud A, et al.: Measured parameters of L-asparaginase experiment at the 28-days time point.2021.\n\nMona Ali Mahmoud A, et al.: Results of histopathological analysis.2021.\n\nAssar MAM, et al.: ARRIVE Guidelines checklist for ‘Effects of asparaginases and L-carnitine on Western-diet-induced hepatosteatosis in mice’. figshare. 2022." }	[ { "id": "121811", "date": "07 Feb 2022", "name": "Matías A. Avila", "expertise": [ "Reviewer Expertise Experimental hepatology", "liver injury/regeneration", "liver fibrosis", "carcinogenesis", "metabolism", "gene expression", "epigenetics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study Mahmoud Assar and coworkers evaluate the hepatotoxicity of two asparaginases formulations in mouse models of fatty liver disease. This is a meaningful study, given the use of asparaginases for the treatment of acute lymphoblastic leukemia (ALL) and the increased liver toxicity found in the clinical setting when patients with hepatopathies, or elderly individuals are treated. Fatty liver develops with age in the general population, therefore the chosen mouse model of fatty liver disease is very relevant. The authors also test the potential protective effects of L-carnitine. The major findings indicate enhanced asparaginase toxicity in mice with fatty liver, mainly for the more stable asparaginase formulation Oncaspar. L-carnitine showed no protective effect. This is a relevant work providing useful information with clinical relevance. I only have a few suggestions for the authors that if addressed could improve their report.\nIt would be helpful to justify in the Methods section how the doses and administration regimes of Oncaspar and L-asparaginase (Spectrila) to mice were selected.\n\nThe composition of the Western diet should be detailed.\n\nWere there signs of inflammation in the livers of mice fed this Western diet? If so, did L-asparaginases administration have an effect on inflammatory parameters?\n\nCould you please discuss why Oncaspar seemed to reduce cholesterol levels increased by Western diet feeding?\n\nCan you please add a short comment on the potential relevance of the major findings of this study regarding the application of L-asparaginase formulations in patients with ALL.\n\nPlease provide information about the size of space bars that are given in the different figures in the legends to the figures.\n\nCan you provide a list of abbreviations (NL, NL L, FL L) because this would increase the readability of the paper.\n\nPlease change “NaCL” for NaCl in all figures.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "161777", "date": "05 Jul 2023", "name": "Ruchi Bansal", "expertise": [ "Reviewer Expertise Fatty liver diseases", "animal models", "diagnosis and therapeutics", "single-cell omics", "hepatic steatosis", "liver inflammation", "liver fibrosis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and relevant study where the authors have studied the effects of Asparaginases and L-carnitine on western diet-induced hepatosteatosis. The authors have demonstrated asparaginase-induced hepatotoxic effects and that L-carnitine did not show any protective effect on the toxicity of asparaginases. I have several suggestions and questions which if implemented can improve the quality of the work.\n1. In the introduction, Spectrila could be included as a recombinant \"native L-asparaginase isolated from Escherichia coli\" similar to Oncaspar.\n2. Experimental design:\nThe animal study experimental design should be improved. The diet composition and the administration route (e.g., not available for Spectrila), etc. should be provided. When L-C (300 mg/kg body weight) was injected (before or after Spectrila) is not clear.\n\nIt is unclear why the authors used young animals in this study. I would have expected the use of older animals to mimic more elderly age groups.\n\nIn the first study, different asparaginase was investigated as compared to the second animal study. Is this first short-term study used to guide the second animal study? If yes, then why did the the authors change female mice to male mice?\n\nIn the first animal study, only 5 mice are used while in the second animal study, 10 mice per group are used. Why is that the case?\n\nIn my opinion, the choice of model should be motivated by the clinical situation. Is there a way to justify their use of animal models motivated by the clinical situation?\n\n3. Results:\nIn my opinion, having results split into figures and tables is confusing. I propose to include the clinical chemistry measurement results in the table and other results i.e., body weight, WBCs, staining, etc. in the figure.\n\nIn the first animal study, I would have expected concentration-dependent effects, however the effects are not concentration-dependent. Why this is the case? Can the authors explain these observations?\n\nSome of the error bars are missing in Figure 1?\n\nBody weight changes presented from day 0 or from the day of the start of the feeding time?\n\nIs the change in body weight reflected in food consumption? Why did the animals lose weight? Is the organ weight data available?\n\nOil-red-o staining results should be included as supplementary or in Figure 2. Error bars are missing for some of the bars.\n\nIt is not clear what the authors mean by degenerative changes and how are they assessed.\n\nIn Figure 3, it is not clear if the data is presented from a 4-day or 28-day time point. this should be clarified. Also, the n number should be mentioned in all the figures and table legends.\n\nIt is surprising that the control (healthy and untreated/vehicle-treated FL) groups are missing but the authors mentioned under table 3 that the significance was calculated versus the FL+NaCl group while it is not mentioned in the experimental design in Figure 3A. These groups should be included for a fair comparison.\n\nPlease see my comments for the first animal study related to body weights and organ weights.\n\nIn general, it would have been fair to compare in one study (containing male and female mice) the effect of recombinant L-asparaginase (Spectrila) and pegaspargase (Oncaspar) with and without L-Carnithine. It is not entirely clear why the studies were performed as they were performed here.\n\n4. Discussion: In the discussion section, it is good to motivate the study design i.e., the choice of the model, the choice of different asparaginases, the choice of male versus female, doses, and administration route with the clinical situation or treatment regimen. Also if the pre-existing conditions such as hepatopathies are comparable and if the toxic effects of asparaginases are comparable to what is observed in clinical settings. Finally, how this study can improve the clinical trials and/or could be useful should be elaborated.\n\nIs the work 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": "182977", "date": "24 Jul 2023", "name": "Olamide B Adelusi", "expertise": [ "Reviewer Expertise Liver toxicology", "acetaminophen induced liver injury", "liver regeneration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, the authors aimed to investigate the hepatotoxicity of two asparaginases in normal and western diet fed mice, as well as L-carnitine as a potential antidote to asparaginase induced hepatotoxicity. The work is interesting with potential clinical relevance, however, several points should be addressed:\nThe methods section is missing some details such as when L-carnitine was given and the route of administration of L-asparaginase.\n\nHow were the doses of Oncaspar, L-asparaginase and L-carnitine arrived at? Are these human relevant doses? An explanation would be helpful.\n\nHow was the number of animals for the different experiments decided? For the Oncaspar study, 6 mice per group were used while for L-asparaginase a much larger number (20 mice per group). Is one of the experiments statistically insufficiently powered or overpowered?\n\nWhy were some experiments performed using Oncaspar and others using L-asparaginase?\n\nIs the western diet model reiterative of what is observed clinically? Why not use aged mice since the elderly are the group most at risk of asparaginase toxicity in clinical settings?\n\nWhy was 4 days after the treatments selected for endpoint measurement? How long after the treatments would the effects of Oncaspar and L-asparaginase be expected to persist? It is possible that the liver has started to recover from the insult in the days since the last treatment. Perhaps measuring liver injury endpoints closer to the end of treatment might show more pronounced effects.\n\nTo the previous point, the effect of Oncaspar and L-asparaginase with or without L-carnitine on liver regeneration should be investigated. Immunostaining for cell cycle proteins such as PCNA or Ki67 is one way to do this.\n\nLiver to body weight ratio would be a more relevant parameter in this case than gross body weight.\n\nMany graphs are missing error bars e.g. figure 1C, 2B, 2C, etc. Also, the number of mice per group should be indicated in the figure legends.\n\nSome of the measurements such as renal and pancreatic injury parameters can be removed or moved to supplements as they do not contribute to the narrative.\n\nIn figure 2, the oil red o images should be included.\n\nIn figure 3, it is not clear if the results are from day 4 or day 28.\n\nWhile the authors showed some clinical and histological changes with Oncaspar and L-asparaginase treatment, these are very small in magnitude and may not be clinically significant. Some discussion of what is observed in patients is necessary to contextualize these findings.\n\nGiven the relatively small increase in steatosis and liver injury in FL + L-asparaginase group compared to FL + NaCl, it is difficult to tease out any protective effect of L-carnitine. A more robust toxicity phenotype is necessary to give conclusive evidence on the effect of L-carnitine on L-asparaginase induced hepatotoxicity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-128
https://f1000research.com/articles/10-699/v1	30 Jul 21	{ "type": "Research Article", "title": "Accumulation of essential (copper, iron, zinc) and non-essential (lead, cadmium) heavy metals in Caulerpa racemosa, sea water, and marine sediments of Bintan Island, Indonesia", "authors": [ "Tengku Said Raza’i", ". Thamrin", ". Nofrizal", "Viktor Amrifo", "Hilfi Pardi", "Imam Pangestiansyah Putra", "Try Febrianto", "Aidil Fadhli Ilhamdy", ". Thamrin", ". Nofrizal", "Viktor Amrifo", "Imam Pangestiansyah Putra", "Try Febrianto", "Aidil Fadhli Ilhamdy" ], "abstract": "Background: Heavy metals are materials naturally occurring in nature and increase with a rise in human activity. Ex-mining areas and domestic waste from human settlements are sources of heavy metal contamination that enter and pollute water, which then accumulates in various organisms including the Caulerpa racemosa community. The accumulation of heavy metals in C. racemosa has a wide impact on the food chain in aquatic ecosystems and humans because this alga is a consumptive commodity.\n\nMethods: Sampling of C. racemosa was carried out at seven sites on Bintan Island, Indonesia covering the eastern (Teluk Bakau, Beralas Pasir, Malang Rapat), northern (Berakit and Pengudang), western (Sakera), and southern parts (Tg. Siambang). Sampling was carried out during different monsoons, and heavy metals in water and sediment samples were measured to determine the heavy metal concentration. Heavy metals were analyzed by a spectrophotometric method using Atomic Absorption Spectrophotometry.\n\nResults: The results showed that heavy metal concentrations fluctuate according to changes in the wind season, which carry currents and spread pollutants into the water. The concentration of metal in the water is also from anthropogenic activities. The heavy metal content of cadmium (Cd), lead (Pb), copper (Cu), iron (Fe), and zinc (Zn) in C. racemosa is high in locations close to settlements. Meanwhile, in seawater samples, Fe and Zn metals have the highest concentrations compared to others. Conclusions: Ex-bauxite mines are a source of Fe and Zn metal contamination in the environment, especially at Tg. Siambang. The levels of these heavy metals in the sediment are also high, as surface particle deposits accumulate at the bottom of the sediment. In general, the levels of heavy metals Cd, Pb, Cu, Fe, and Zn increase in the northern monsoon because the dynamics of the water transport greater heavy metal pollution.", "keywords": [ "Heavy metal", "Caulerpa racemosa", "Bintan Island", "Atomic Absorption Spectrophotometry" ], "content": "Introduction\n\nHeavy metals are materials naturally contained in nature. These metals increase along with a rise in human activity and become a threat to environmental pollution. The increase in heavy metals is caused by mining,1 agriculture and industry2 activities, as well as anthropogenic processes from residential and household domestic waste.3,4 Therefore, increased coastal activity leads to a rise in the content of these metals up to the quality standard threshold.5\n\nHeavy metal particles that accumulate in the environment affect aquatic ecosystems. The contamination is distributed in the water column and accumulates in organisms over a prolonged period, affecting the food chain.5 Briffa et al. (2020) stated that heavy metal contamination in organisms affect biological functions and cause various toxic effects in the long term.6 According to Liu et al. (2020) and Apiratikul et al. (2004), these metals are generally dominated by cadmium (Cd), lead (Pb), copper (Cu), iron (Fe), and zinc (Zn).1,7–9\n\nCadmium (Cd) and lead (Pb) are non-essential heavy metals whose function in the human body are unknown. These metals are categorized as carcinogenic (or mixed) agents for humans by the International Agency for Research on Cancer. Exposure to Cd and Pb causes cancer or infection in human organs such as the urinary tract, reproductive tract, central nervous system, respiratory system,10,11 they cause kidney problems, and increase blood pressure.11 The content of these heavy metals are measured in blood, urine, hair, nail, and saliva samples and are characterized by low levels of excretion.12 Furthermore, excess exposure to Cd and Pb is lethal when entering mammalian cells and they accumulate high concentrations in the cytoplasmic space and nucleus.13,14 The maximum limits of Cd and Pb metals in the human body are 0.005 mg/L and 0.015 mg/L, respectively.15 Therefore, due to the high level of toxicity of heavy metals, serious monitoring of the accumulation of Cd and Pb is needed.\n\nMeanwhile, copper (Cu), zinc (Zn), and iron (Fe) are essential heavy metals, with a certain amount needed in the human body. However, when the amount exceeds the maximum limit, it becomes poisonous and very dangerous to health. Cu is considered a carcinogen metal because it causes damage to DNA. Meanwhile, Cu2+ interacts with lipid hydroperoxides to form malondialdehyde and 4-hydroxynonenal, which are considered carcinogens and cause DNA and tissue damage.16,17 Excessive accumulation of Zn metal causes depletion of Cu2+ in cells and decreases superoxide dismutase (SOD) and cytochrome C oxidase levels by increasing cholesterol levels. Furthermore, it causes cardiac dysfunction with impaired iron mobilization, inhibits the enzymes SOD, peroxidase, and catalase, thereby rapidly increasing the concentration of superoxide free radicals and oxidative stress. Excess Zn also causes severe damage to the cell wall and DNA while increasing gene mutations.18 Fe metal is also one of the important elements in the body with a tightly controlled concentration. Excess Fe is caused by several conditions such as frequent transfusions, exploitation of iron consumption (as a supplement), and chronic hepatitis. It triggers the formation of free radicals, thereby causing severe complications such as mental retardation, Alzheimer's, multiple sclerosis, reproductive system dysfunction, heart problems, liver cirrhosis, liver cancer, hepatitis, and metabolic dysfunction.19 The maximum limits for the essential heavy metals Cu, Zn, and Fe in the human body are 1.3 mg/L, 3 mg/L, 0.2 mg/L, respectively.15 Therefore, it is necessary to monitor the concentration of these heavy metals to prevent them from passing the accumulated threshold.\n\nAquatic ecosystems consist of various types of biota including the macroalgae community such as Caulerpa racemosa. Heavy metal pollution in the aquatic environment also impacts the accumulation of these metals in macroalgae.7 Caulerpa sp. is a type of algae that is able to absorb Cd, Pb, Cu, Fe and Zn metals in water.20 The absorption ability of Pb,21 Cd 30 g/L,22 Zn and Cu 10 mg/L,23 and Fe 7.63 mg/L24 in water is 0.35 mg/kg. This proves that C. racemosa has the ability to bioaccumulate heavy metals in marine waters.\n\nOn the other hand, C. racemosa has consumptive benefits for coastal communities, especially as a traditional food ingredient. Khandaker et al. (2021) stated that the accumulation of heavy metals in these organisms have the potential to cause various adverse health effects.25 The content of heavy metals contained in C. racemosa in Bintan Island, Indonesia waters is unknown, therefore, it is important to carry out this research in order to determine the impact of its exposure to surrounding communities.\n\n\nMethods\n\nData were collected from seven sites on Bintan Island, Indonesia covering the eastern (Teluk Bakau, Beralas Pasir, Malang Rapat), the northern (Berakit and Pengudang), the western (Sakera), and the southern part (Tg. Siambang). In addition, data was also taken based on seasonal differences to determine the effect of seasonal changes on the existing conditions of C. racemosa on the coast of Bintan Island. The difference in seasons is taken to refer to the difference in the direction of the monsoon, namely the northerly, easterly, southerly and westerly monsoon. Figure 1 shows the distribution of research sites on Bintan Island.\n\nMap of research locations and field data collection, This figure has been reproduced with permission from Apriadi et al., (2018).58\n\nSampling of C. racemosa was carried out using a scuba set (Amscud), 50 × 50 cm2 transect plot, underwater camera (Canon D30 82 '/25m rated depth), GPS (Garmin GPSMAP 78S), Oven (Memmert UN 55), analytical scales (Kern ABJ 220, accuracy level of 0.001 g), Van Dorn sampler, sediment shovel, mortar, aluminium foil, plastic samples, label paper, and boxes. Heavy metal levels were tested by the wet digestion method using a solution of nitrite acid (HNO3), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), and hydrochloric acid (HCl).20 Samples were analyzed using Atomic Abstraction Spectrophotometry (AAS) using the Shimadzu AA-7000.26\n\nThe sample preparation process was carried out by weighing 5 grams of the dry weight of the C. racemosa sample, which had previously been cleaned and dried in an oven at 60°C for 24 hours.27,28 The sample was then crushed with a mortar until homogenous before adding a HNO3 solution and 20 ml of HCl. Furthermore, the solution was placed in a water heater until it reacted and was digested. It was then filtered with Whatman 42 filter paper to determine the readings of Cd, Pb, Cu, Fe, Zn on the AAS tool.\n\nWater and sediment sampling for heavy metal analysis refers to the digestion method.29 Approximately three liters of seawater sample were taken with a Van Dorn sampler and then concentrated by baking at 80°C until the volume reduced to 50 ml. Furthermore, 4 ml of H2SO4, and 10 ml of H2O2 were added to the sample and heated until the oxidation was complete. After cooling the sample, it was filtered with 0.45 μm Whatman 42 filter paper and distilled water was added until it reached a volume of 50 ml.\n\nThe sediment taken in the field was weighed, and as much as 30 g was dried in an oven at 105°C. In addition, approximately 1 g of the sediment was added to 5 ml of HNO3 and heated at 80°C until the volume changed (20–30 ml). The process of adding HNO3 was repeated and it was reheated, after which it was filtered with 0.45 μm Whatman filter paper and distilled water was added until it reached a volume of 50 ml.\n\nThe results of the metal content readings of Cd, Pb, Cu, Fe, Zn were analyzed using IBM SPSS Statistics 22 software with one-way Anova analysis (p <0.05) to determine the concentration of heavy metals at each site based on the season. Furthermore, the level of correlation between metals was also analyzed to determine the influence of each metal being measured.\n\n\nResults and discussion\n\nHeavy metal analysis was distinguished by season to determine the content in C. racemosa and to explain the effect of the season on the water. Those heavy metals measured in the C. racemosa sample consisted of Cd, Pb, Cu, Fe, and Zn in micrograms per gram (μg/g), with the analysis results shown in Table 1. The concentration of the metals Cd, Pb, Cu, Fe, and Zn were in the range of 0.14–4.83 μg/g, 1.07–47.95 μg/g, 1.14–8.14 μg/g, 62.4–609.7 μg/g, and 12.7–76.5 μg/g with an average concentration of 0.95 ± 1.15 μg/g, 14.17 ± 10.98 μg/g, 3.07 ± 2.16 μg/g, 220.58 ± 175.16 μg/g, and 35.25 ± 16.54 μg/g, respectively.\n\nCd = cadmium, Pb = lead, Cu = copper, Fe = iron, Zn = zinc.\n\n* The residential areas: Ml. Rapat, Tl. Bakau, B. Pasir, Pengudang, Sakera.\n\n The ex-bauxsite mine: Tg. Siambang.\n\nResearch by Khaled et al. (2014) obtained the results of the content of several metals in macroalgae, namely Cd (0.26 μg/g), Cu (6.0 μg/g), Fe (177.79 μg/g), Pb (41.16 μg/g) and Zn (59.13 μg/g).30 Yulianto et al. (2018) showed that the concentration of Cd metal in seaweed ranged from 0.018 to 0.87 μg/g.31 Meanwhile, research by Llanos and Dalawampu (2017) on a C. racemosa sample stated that the concentration of heavy metals such as Cd, Pb, and Zn was in the range of 0.21–0.29 μg/g, 2.61–7.23 μg/g and 6.03–7.23 μg/g, respectively.32 Intawongse et al. (2018) research in C. racemosa obtained metal concentrations of Pb (0.43 μg/g), Cd (0.79 μg/g), Cu (1.55 μg/g), Fe (311 μg/g), and Zn (13.4 μg/g).33 The results of the above studies indicate that metal levels in sea water fluctuate according to the conditions, with the concentrations influenced by other effects, especially anthropogenic activities.\n\nTable 1 shows an increasing trend of heavy metal concentrations in residential areas and as a result of former bauxite mining activities. As is shown from seven observations, the waters of Malang Rapat, Teluk Bakau, Berakit, Pengudang, and Sakera. The observation station in Tg Siambang waters is a former bauxite mining area whose effects are now affecting the surrounding aquatic environment.\n\nThe metal concentrations of Cd, Pb, Cu, and Fe in the ex-mining area have a higher value than in other areas. For example, the concentration of Cd in ex-mining areas is between 2.17–4.83 μg/g, while in other areas, it is lower between 0.14–1.12 μg/g. Similarly, the levels of Pb, Cu, and Fe increased along with a rise in the intensity of community activities.\n\nRehman et al. (2013) compared the levels of several metals in residential areas and found that the Zn, Fe, Cu and Pb content reached 199.94 μg/g, 195.62 μg/g, 9.18 μg/g, and 13.09 μg/g, respectively.34 According to Vongdala et al. (2019) and Agbeshie et al., the concentrations of Fe, Pb, Zn, and Cd at domestic waste disposal sites tend to experience a significant increase.35,36 Kinuthia et al. (2020) stated that an increase in heavy metal concentration positively correlates with the intensity of residential spots.37 This is in line with the results of the study, which identified an increase in the levels of heavy metals Cd, Pb, Cu, Fe, and Zn. Furthermore, this activity causes metal accumulation in C. racemosa to be higher with a greater effect.\n\nHeavy metal concentrations in C. racemosa samples were also differentiated according to the wind season, as shown in Table 1 and Figure 2. The aim was to determine the effect of the season on changes in the concentration of heavy metals that accumulated in C. racemosa. The concentration of Cd, Pb, Cu, and Fe metals increased in seasons with high wave dynamics and currents (Northerly monsoon). The heavy metals that accumulate in C. racemosa are lower in the east-west monsoon (Easterly–Westerly monsoon) than in the north (Northerly monsoon). The Cd metal content in the east-west monsoon, namely 0.71 ± 0.67 and 1.09 ± 1.27 μg/g increased to 1.20 ± 1.63 μg/g in the northern monsoon. Furthermore, the concentration of Pb in the east-west monsoon, namely 2.70 ± 2.37 and 13.29 ± 6.07 μg/g increased in the northern monsoon to 22.98 ± 11.92 μg/g. Additionally, the concentration of Cu in the east-west monsoon was 2.92 ± 2.3 μg/g compared to 3.40 ± 2.05 μg/g in the northern monsoon. Fe content in the east-west monsoon, namely 209.63 ±208.31 and 240.34 ± 190.19 μg/g increased to 254.65 ± 201.36 μg/g in the north monsoon. Zn metal in the east-west monsoon increased from 24.34 ± 7.69 and 34.08 ± 8.73 μg/g to 51.12 ± 22.94 μg/g in the north monsoon. These results indicate that all the tested metals increased in the northern season.\n\nTalwar et al. (2014) carried out research related to differences in heavy metal concentrations by season. The results showed that Pb, Cr, and Cu tend to increase during the rainy season by 0.03–0.8 ppm, 0.01–0.4 ppm, and 0.02–0.7 ppm, respectively.38 According to research by Mondol et al. (2011), the highest concentration of heavy metals in marine plants is during the wet season, where Pb, Cr, and Cu metals range from 6.18 to 14.91 μg/g, 19.65–19.83 μg/g, and 23.88–33.16 μg/g.30 Zhang et al. (2017) stated that the rainy season has the highest effect on the increase in heavy metals.39 This condition is similar to the results found at this research location, where the concentration of heavy metals tends to increase in the northern monsoon due to the effect of current and wave dynamics, which are the driving force for marine dynamics that carry and spread pollutants in waters.40\n\nKoropitan and Cordova (2017) stated that flow is a component of heavy metal transport from a river transported into open water, thereby increasing its value in the aquatic ecosystem.41 Budiyanto and Lestari (2017) examined the difference between metals in the eastern (March) and the southern seasons (June).42 The results showed an increase in Cd, Cu, Pb and Zn metals from 1.18 μg/g to 1.74 μg/g, 94.8 μg/g to 157.0 μg/g, 41.7 μg/g to 89.4 μg/g, and 503.0 μg/g to 1270 μg/g, respectively. These results clearly show that changes in environmental conditions, especially wind direction, dynamics of currents and waves, as well as season, were factors that affected heavy metal concentrations in nature.\n\nThe one-way Anova test (Table 2) showed that the concentration of Pb in C. racemosa in the northern monsoon was significantly different compared to other seasons (f = 0.051). Meanwhile, the Cu, Fe, Zn and Cd results were not significantly different between seasons with f values of 0.722, 0.481, 0.121, and 0.493, respectively. Therefore, it can be concluded that the fluctuating season conditions do not significantly affect changes in metal concentrations. The levels of correlation between Cd, Pb, Cu, Fe, and Zn metals are shown in Table 3.\n\nCd = cadmium, Pb = lead, Cu = copper, Fe = iron, Zn = zinc.\n\n Correlation is significant at the 0.01 level (2-tailed).\n\nThe results of metal analysis in C. racemosa showed a strong correlation level in Pb with Cd, Cu with Cd, Fe with Cd, Cu with Pb, Fe with Pb, Zn with Pb, Fe with Cu, and Zn with Cu. Research by Wang and Liu (2004) found a correlation between Pb and Cd, Cu with Pb, and Zn with Pb metals.43 Furthermore, research by Khaled et al. (2014) showed a positive correlation value between metals Cu and Cd, Fe with Cd, metal Cd with Pb, metal Cu with Fe, Cu with Pb, Cu with Zn, and Fe with Pb.44 Harikumar et al. (2010) also obtained a positive correlation value between heavy metals Cu and Pb.45 Mourad and El-Azim's research (2019) on macroalgae showed a positive correlation between Cu and Cd, Cu with Pb, Fe with Pb, Zn with Pb, Fe with Cu, and Zn with Cu.46 The concentrations of metals in C. racemosa correlate with each other, which indicates that an increase in the levels of one metal affects others.\n\nThe concentration of heavy metals in the tested water samples fluctuated according to seasonal conditions. It was found that the highest average concentrations of Cd, Pb, Cu and Fe were in the eastern monsoon, namely 0.0013 ± 0.00056 mg/l, 0.0010 ± 0.00048 mg/l, 0.0010 ± 0.00074 mg/l, and 0.0013 ± 0.00092 mg/l, respectively. Meanwhile, Zn metal was the highest in the western monsoon with an average of 0.0240 ± 0.0054 mg/l, as shown in Table 4.\n\nCd = cadmium, Pb = lead, Cu = copper, Fe = iron, Zn = zinc.\n\n* The residential areas: Malang Rapat, Teluk Bakau, Beralas Pasir, Pengudang, Sakera.\n\n** The ex-bauxsite mine: Tg. Siambang.\n\nSeveral studies stated that in marine waters, Pb metal concentrations ranged from 0.0059–1.07 mg/l,47 Cadmium (Cd) ranged from 0.01–0.03 mg/l,48 Cu ranged from 0.0472–0.0725 mg/l, Zn ranged from 32.59–45.79 mg/l,49 and Fe ranged from 0.17–0.45 mg/l.50 In general, the concentrations of heavy metals in the water samples at the research locations were similar to previous studies, though the levels of Fe and Zn were categorized as high in this research.\n\nBazzi (2014) stated that anthropogenic activity is a determining factor for high Zn and Fe metal contamination in waters.51 Furthermore, Sun et al. (2020) emphasized that heavy metal levels are influenced by changes in salinity, pH, and biological activity, they tend to increase at the location of bays and estuaries that are close to residential activities.52 Zn and Fe metal contamination in the research location is influenced by community activities, especially settlements that produce various types of waste. In addition, the open land of former bauxite mining on Bintan Island is a source of contamination of these metals in the waters. According to Rezaei et al. (2019) and Ismail et al. (2019), bauxite waste is a source of Fe and Zn metal contamination in the environment.53,54 It is one of the causes of the high concentration of Zn and Fe metals in the research location.\n\nHeavy metals that enter the water accumulate at the bottom and affect the metal composition of the sediments. According to preliminary studies, the highest content of heavy metal Cd in sediments occurred in the westerly monsoon with an average of 0.0289 ± 0.037 μg/g, while the northerly monsoon produced strong current dynamics of 0.0045 ± 0.001 μg/g. The highest Pb content was in the easterly monsoon with an average of 0.0449 ± 0.013 μg/g, the highest Cu and Fe was in the west season with an average of 0.0321 ± 0.024 μg/g and 18.43 ± 5.25 μg/g, respectively. However, the heavy metal Zn actually increased during the northerly monsoon with an average value of 11.63 ± 14.041 μg/g, as shown in Table 5.\n\nCd = cadmium, Pb = lead, Cu = copper, Fe = iron, Zn = zinc.\n\n* The residential areas: Malang Rapat, Teluk Bakau, Beralas Pasir, Pengudang, Sakera.\n\n** The ex-bauxsite mine: Tg. Siambang.\n\nThe concentration of heavy metals in sediments decreases and increases in the northern monsoon and east to west monsoons, respectively. The stirring of sediment that occurs in the northern monsoon is a factor in the difference in metal concentrations between seasons. The water currents stir the sediment in seasons with hydrodynamic conditions, therefore, the heavy metals contained are dispersed more quickly. Meanwhile, in other seasons, the dynamics are smaller and support the deposition of metal particles that enter the waters. This affects the accumulation of heavy metals in the water to the bottom sediments. Wardani et al. (2020) stated that heavy metals are transported by currents where the flow conditions differ in strength in accordance with the season.55 Wisha et al. (2018) and Hamuna and Tanjung (2021) stated that the distribution of heavy metals in the sea is influenced by several water inputs and dynamics, topography, wind patterns, movement and surface current circulation.56,57\n\n\nConclusions\n\nIn conclusion, the concentration of Cd, Pb, Cu, Fe and Zn in C. racemosa ranged from 0.14–4.83 μg/g, 1.07–47.95 μg/g, 1.14–8.14 μg/g, 62.4–609.7 μg/g, and 12.7–76.5 μg/g with averages of 0.95 ± 1.15 μg/g, 14.17 ± 10.98 μg/g, 3.07 ± 2.16 μg/g, 220.58 ± 175.16 μg/g, and 35.25 ± 16.54 μg/g, respectively. Heavy metal fluctuations are closely related to seasonal changes, where in the east-west monsoon (Easterly–Westerly monsoon), those that accumulate in C. racemosa are lower than in the north monsoon (Northerly monsoon). However, based on the one-way Anova analysis, only Pb was significantly different between seasons, while Cd, Cu, Fe, and Zn were insignificant. The correlation between metals showed a positive relationship, which indicates that the increase in one metal affect others.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nThe authors are grateful to the government of Teluk Bakau Village, Malang Meeting, Berakit, Beralas Pasir, Pengudang, Sakera, and Dompak, as well as the survey team for providing access to data collection in the field on Caulerpa racemosa.\n\n\nReferences\n\nLiu R, Xu Y, Zhang J, et al.: Effects of heavy metal pollution on farmland soils and crops: A case study of the Xiaoqinling Gold Belt, China. China Geol. 2020; 3: 402–410. Publisher Full Text\n\nArmah FA, Quansah R, Luginaah I: A Systematic Review of Heavy Metals of Anthropogenic Origin in Environmental Media and Biota in the Context of Gold Mining in Ghana. Int. Sch. Res. Not. 2014; 2014: 1–37. 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PubMed Abstract \| Publisher Full Text\n\nNindyapuspa A, Ni’Am AC: Distribution of heavy metals (Cu and Fe) in sea water of Gresik coastal area. E3S Web Conf. 2018, 2017–2019; 31. Publisher Full Text\n\nBazzi AO: Heavy metals in seawater, sediments and marine organisms in the Gulf of Chabahar, Oman Sea. J. Oceanogr. Mar. Sci. 2014; 5: 20–29. Publisher Full Text\n\nSun X, Li BS, Liu XL, et al.: Spatial Variations and Potential Risks of Heavy Metals in Seawater, Sediments, and Living Organisms in Jiuzhen Bay, China. J. Chem. 2020; (2020). Publisher Full Text\n\nRezaei A, Hassani H, Fard Mousavi SB, et al.: Evaluation of Heavy Metals Concentration in Jajarm Bauxite Deposit in Northeast of Iran Using Environmental Pollution Indices. Malaysian J. Geosci. 2019; 3: 12–20. Publisher Full Text\n\nIsmail SNS, Abidin EZ, Praveena SM, et al.: Heavy metals in soil of the tropical climate bauxite mining area in Malaysia. J. Phys. Sci. 2018; 29: 7–14. Publisher Full Text\n\nWardani NK, Prartono T, Sulistiono: Sediments quality based on geo-accumulation index in heavy metals (Pb, cu, and cd) of cengkok coastal waters, banten bay. J. Pendidik. IPA Indones. 2020; 9: 574–582. Publisher Full Text\n\nWisha UJ, Heriati A, Ramdhan M, et al.: Spatial Distribution of Dissolved Heavy Metals (Hg, Cd, Cu, Pb, Zn) on the Surface Waters of Pare Bay, South Sulawesi. ILMU Kelaut. Indones. J. Mar. Sci. 2019; 23: 199. Publisher Full Text\n\nHamuna B, Tanjung RHR: Heavy metal content and spatial distribution to determine the water pollution index in depapre waters, Papua, Indonesia. Curr. Appl. Sci. Technol. 2021; 21: 1–11.\n\nApriadi T, Pratama G, Putra RD, et al.: Comparative study on the fish diversity from natural and bauxite postmining in wetland system of Bintan Island, Indonesia. Biodiversitas. 2018; 19: 967–973. Publisher Full Text" }	[ { "id": "99666", "date": "17 Nov 2021", "name": "Ong Meng Chuan", "expertise": [ "Reviewer Expertise marine pollution", "heavy metals pollution" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMethods:\nProvide citation for the acid digestion used in this study.\n\nWhat standard reference material was used in this study? What is the recovery test?\n\nNo citation provided for the water analysis and sediment analysis.\n\nNo detailed digestion method provided.\n\nResults & discussion:\nI would suggest that the data is presented in graphs for the comparison between.\n\nI'm not sure whether the data presented is acceptable or not since no recovery percentage is provided.\n\nAre the authors Khaled et al. also studying the same sp - because the authors only mention macroalgae.\n\nMore explanation about why the ex-bauxite mining area is high with metals content.\n\nFor the correlation of metals, the authors only compare the data with other investigators - should include what the correlation means? Any correlation with size?\n\nThe concentration of metals in sediment is very low. But I can't say whether the data is acceptable or not, since no recovery percentage is provided. Please check the data.\n\nSince the authors study on organisms, water and sediment, what is the correlation between these three samples? No explanation is provided. Suggest to run PCA analysis.\n\nConclusion:\nThe conclusion is only based on organisms - how about water and sediment? Please rewrite.\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? No", "responses": [ { "c_id": "7711", "date": "01 Feb 2022", "name": "hilfi pardi", "role": "Author Response", "response": "Provide citation for the acid digestion used in this study. Response: Added: V. van Ginneken and E. de Vries, Seaweeds as Biomonitoring System for Heavy Metal (HM) Accumulation and Contamination of Our Oceans, Am. J. Plant Science. 09 (2018), pp. 1514–1530. The analytical procedures must be validated, one of which is using the recovery test Response: The analytical procedures must be validated, one of which is using the recovery test No citation provided for the water analysis and sediment analysis. Response: S. Shanbehzadeh, M. Vahid Dastjerdi, A. Hassanzadeh and T. Kiyanizadeh, Heavy metals in water and sediment: A case study of Tembi River, J. Environ. Public Health 2014 (2014), . No detailed digestion method provided. Response: It can be seen in the following references: V. van Ginneken and E. de Vries, Seaweeds as Biomonitoring System for Heavy Metal (HM) Accumulation and Contamination of Our Oceans, Am. J. Plant Sci. 09 (2018), pp. 1514–1530. For the correlation of metals, the authors only compare the data with other investigators - should include what the correlation means? Any correlation with size? Response: In this study, the PCA test and the correlation between heavy metals contained in water, Caulerpa, and sediment were carried out with water quality parameters. References Khaled et al. (2014) less relevant Response: Modified and refers to Ginneken and Vries (2018). Why is the content of heavy metals in the ex-bauxite area higher? Response: It is explained in the chapter on the results and discussion of the sub-chapter “Heavy metals in Caulerva racemosa samples” the 5th paragraph. Since the authors study on organisms, water and sediment, what is the correlation between these three samples? No explanation is provided. Suggest to run PCA analysis. Response: Added a new chapter “PCa and Correlation of heavy metal” in the results and discussion chapter. Correlation Test Results: - Between water quality and metal concentration in Cauleroa - Between water quality and metal concentration in water - Between water quality and metal concentration in sediment Response: Listed in Tables 4, 5, and 6 of the Appendix. I would suggest that the data is presented in graphs for the comparison between. Response: Results are listed in Figures 3, 4 and 5. Pca analysis includes the scatterplot Response: Results listed on Figure 6. The conclusion is only based on organisms - how about water and sediment? Please rewrite. Response: Was added to the conclusion for metals in water and sediment." } ] } ]	1	https://f1000research.com/articles/10-699
https://f1000research.com/articles/10-1085/v1	25 Oct 21	{ "type": "Research Article", "title": "The benefits of mindfulness in mental healthcare professionals", "authors": [ "Tayler Watson", "Owen Walker", "Robin Cann", "Ashwin K Varghese", "Tayler Watson", "Owen Walker", "Robin Cann" ], "abstract": "Background: Burnout is a widely reported syndrome consisting of emotional exhaustion, depersonalization, and a lowered sense of accomplishment. Mindfulness practices have been shown to be useful in lowering distress and burnout in clinical and non-clinical cohorts. Our aim was to explore the potential personal and occupational benefits of a structured mindfulness intervention on a cohort of mental health professionals. A mixed-methods approach was utilised in order to enhance the exploratory power of the study. Methods: We conducted a pilot study involving healthcare practitioners employed at a community outpatient mental health clinic. As a pilot, we relied on a single group and implemented a quasi-experimental, simultaneous mixed methods design by incorporating both quantitative pre- and post- testing alongside written qualitative post-test responses. Results: Analysis of the data demonstrated a significant difference between overall mindfulness when comparing post-test (mean=140.8, standard deviation=18.9) with pre-test data (mean=128.3, standard deviation=28.6). Participants also showed a statistically significant difference in three of the subscales: observation, describing, and non-reactivity. A moderate effect size was seen for each of the above differences. Analysis of the qualitative data revealed a range of potential themes which may be used to explain the differences exhibited across participants’ personal and professional lives, which can be grouped into two thematic overarching groups: emotional reactivity and listening/communicating. Conclusions: The results of this pilot study indicate that a structured, six-week mindfulness program has the potential to benefit clinicians, personally by reducing emotional reactivity and professionally by promoting deep listening and communication.", "keywords": [ "Mindfulness", "Mental Health", "Burnout", "Psychiatry" ], "content": "Introduction\n\nProposed by Maslach et al. in 1986, burnout is now widely accepted as consisting of emotional exhaustion, depersonalization and a lowered sense of accomplishment, contributing to an overall reduction in psychological wellbeing.1 High levels of burnout have been recorded across industries2–4 with common etiological themes related to workload demands, low occupational satisfaction and psychosocial stressors.5–7 Some reports suggest up to 80% of physicians meet criteria for burnout,8 however more conservative estimates suggest around half of physician’s experience burnout.9 Up to 78% of psychiatrists and 21% of psychiatry residents are experiencing burnout at any one time.10,11 Around half of family physicians report high levels of emotional exhaustion and depersonalization, and more than two thirds of young oncologists report burnout.12,13 Two thirds of mental health clinicians including psychologists, social workers and nurses, also report distress.14 Burnout is associated with major depression, poor patient outcomes and more negative feelings towards patients.15–17 Physician burnout also results in significant economic loss.18 By contrast, psychiatry is well placed to drive positive change.19 It is clear that burnout is a key issue facing the medical and psychosocial professions. While the existence of the phenomenon is well understood, attempts to rectify the problem remain scarce. Interventions would need to be easily administered and suitably scalable. Mindfulness represents a promising avenue for exploration, given the ease with which it can be taught and maintained.\n\nMindfulness can be defined as “paying attention to present moment experiences with openness and curiosity and a willingness to be with what is” (UCLA health). Operational definitions distinguish two components of mindfulness: self-regulation of attention and orientation to the present moment characterized by curiosity, openness and acceptance.20 A range of mindfulness practices and techniques have been developed and applied for use in both mental and physical illnesses.21 One of these practices, Mindfulness Based Stress Reduction (MBSR), demonstrates efficacy in reducing stress, depression, anxiety and negative ruminations.22–24 MBSR delivered to therapists in training enhanced self-compassion and positive affect.25 When applied to physicians, MBSR may reduce burnout, enhance resilience and improve patient care.26–28 Mindfulness has also been correlated with neurophysiological and neurobiological changes in key brain regions associated with the observed phenomenology.29\n\nThis preliminary pilot study aims to explore the benefits of a mindfulness intervention on mental health practitioners. Mixed methods approaches have been shown to be an effective way to allow for a more robust exploration of a phenomenon than each individual method used in isolation.30 Collecting both types of data can greatly assist in the exploratory nature of the study, with qualitative information used to further explore, as well as potentially explain, the quantitative analysis.\n\n\nMethods\n\nThis pilot study employed a single group quasi-experimental, simultaneous mixed-methods design by utilizing quantitative pre and post testing paired with a written qualitative post-assessment. The qualitative component of this study used a descriptive phenomenological method.\n\n13 healthcare practitioners employed at a community outpatient mental health clinic in Amherst, Nova Scotia were recruited for the study in October 2016 (Table 1). Recruitment was completed via email and word-of-mouth discussion. Interested clinicians were then asked to refer colleagues who were not reached via email. Non-probability convenience sampling was used to recruit participants with desired study characteristics and expeditiously collect qualitative data. Snowball sampling increased the potential number of participants. This number of participants was chosen as evidence suggests theoretical saturation occurs at 12 respondents.31,32\n\nThe Mindfulness Awareness Practices (MAPs) program is a six-week, validated protocol delivered by a trained instructor. The intervention has demonstrated usefulness in reducing distress in clinical populations.33,34 Participants completed the MAPs six-week course, in November and December 2016. Week one included a two-hour workshop focussed on a conceptual introduction to mindfulness and psychoeducation. In weeks two to five, participants engaged in weekly didactic and experiential 90-minute workshops learning a range of mindfulness techniques, such as sitting, relational and movement meditation and skills for recognising positive and difficult emotions. Participants were encouraged to complete and record ‘homework’ activities, including guided meditation practice. The program was delivered on-site at an outpatient community mental health clinic during the participants’ regular workday.\n\nThe Five Facet Mindfulness Questionnaire (FFMQ), first proposed in 2006, is a robust, 39-item self-report, Likert type scale measuring five correlates of mindfulness; observing, describing, acting with awareness, non-judging and non-reacting.35,36 The survey’s factor structure is available in the original article.35 The scale exhibits reliable construct validity across facets.37 Examples of statements include “I’m good at finding words for my feelings” and “I watch my feelings without getting lost in them”. Respondents are then asked to rate these statements with five potential options (never or very rarely true, rarely true, sometimes true, often true, very often or always true).\n\nThe Perceived Stress Scale (PSS)10 is an extensively used self-report questionnaire designed to measure individual stress, demonstrating high internal consistency across populations (α= 0.82).38,39 Respondents are asked 10 questions, scored on a Likert scale (never, almost never, sometimes, fairly often or very often). Questions assess the level of perceived stress one has experienced within the last month. For example,”in the last month, how often have you been upset because of something that happened unexpectedly”? The scale is widely available and has been validated in several languages.40–42\n\nParticipants completed handwritten, hardcopy FFMQ and PSS questionnaires prior to the first session (week 1) and post-participation in the protocol (week 6). Qualitative data was obtained using brief, open-ended written survey questions that allowed for narrative-style feedback from participants, completed post-intervention. The questions were developed to allow for rich and open responses in the context of the study design and intervention, and therefore were not based on pre-existing models or surveys. The questions were as follows:\n\n“In what way has the six-week mindfulness training influenced your personal life?”\n\n“In what way has the six-week mindfulness training influenced your clinical practice?”\n\nFFMQ and PSS scores were set as primary outcome measures. Continuous data from the quantitative questionnaires was entered into SPSS (version 24). Paired samples t-tests were then conducted. Statistical significance was assumed at 0.05%. Corresponding Cohen's d values were determined, and effect sizes established. Outcome variables included overall mindfulness, perceived stress and the five FFMQ subscales. Secondary outcome measures sought to explore the relationship between engagement and change in outcome scores, however statistical analysis was impacted by poor response rates.\n\nParticipants were provided with the two questions stated above, and in turn provided the researchers with written responses. Written responses were transcribed to text using Microsoft Word (version 16.53) and some minor substitutions (e.g., replacing shorthand with full words) were made to aid legibility, with no impact to the meaning of the responses. 1% of words were illegible, despite consulting a second reviewer. Thematic analysis was applied to the qualitative data to observe themes that arose across participants’ varying experiences of the program. This allowed the authors to identify, analyse and describe recurrent themes and concepts observed within the dataset. Thematic analysis is a flexible approach that provides a framework for a rich discussion.43 Considering the subjectivity of the research topic, the authors adopted this approach to best reflect the true experience of the participants, and thus enhance trustworthiness.\n\nThe researchers acknowledge their own characteristics in the interpretation of the qualitative data. Interpreting authors were predominantly male from medical backgrounds with special interests in the subject matter, which we recognise may have impacted objectivity.\n\nThe Nova Scotia Health Authority Research Ethics Board granted an exemption from ethics approval as the study is a quality improvement/educational initiative. Verbal, informed consent was obtained from participants prior to commencement for use and publication of study data as all participants were staff of the health authority. The paper forms were stored in a locked cabinet and digital data were stored on password-protected computers.\n\n\nResults\n\nDemographic characteristics of the cohort are displayed in Table 1. Most participants were female and either worked in psychiatry or social work. Table 2 displays statistically significant results of paired sample T-tests, used to measure pre-post change in overall mindfulness and FFMQ subscales. On the FFMQ, there was a statistically significant difference in overall mindfulness after participation in MAPs when compared to before participation (p = 0.007) (Figure 1). Participants also demonstrated statistically significant changes on three subscales of the FFMQ: observation, describing and non-reactivity (Figure 2). There was a statistically significant increase in participants’ ability to observe internal and external experiences when compared to baseline (p = 0.036). Similarly, a statistically significant increase was also demonstrated in participants’ ability to describe and label internal experiences after participation in MAPs than at the time of pre-test (p = 0.042). Finally, participants showed reduced reactivity to internal experiences and enhanced ability to allow the free flow of thought without rejection after participation in the study when compared to baseline (p = 0.019). Moderate effect sizes were demonstrated in the overall FFMQ (Cohen’s d = 0.588), observing (Cohen’s d = 0.452), describing (Cohen’s d = 0.407) and non-reacting (Cohen’s d = 0.693) subscales. The mean number of absences to sessions was 1.2. Reason for absences was not recorded. Three participants missed two or more sessions in total. No statistically significant difference in FFMQ scores was observed in this group (p = 0.98). There were no statistically significant changes on the final two subscales of the FFMQ, awareness and non-judging; as well, no statistically significant changes in the perceived stress scale were observed.\n\nCorresponding effect sizes presented as Cohen's d values.\n\nError bars represent standard errors (SEM). FFMQ: Five Facet Mindfulness Questionnaire. MAPs: Mindfulness Awareness Practices.\n\nError bars represent standard errors (SEM). FFMQ: Five Facet Mindfulness Questionnaire. MAPs: Mindfulness Awareness Practices.\n\nThe responses for each of the two questions were initially analysed separately, with a range of sub-themes identified. Positive effects in participants’ personal life and clinical practice were noted.\n\nA common response amongst participants was the change in their relationship with their emotions, particularly in their capacity to identify, observe, and subsequently regulate them, as evidenced by the following examples: “Better able to notice emotions”, “Increased emotional regulation”, “Checking in more often with feelings and sensations”, “making room and allowing”, “awareness of what I am experiencing in the moment”, “I am less emotionally reactive”, “I am more aware of my emotions and in better control of them”.\n\nTolerance of uncertainty was another common benefit that arose. Some participants indicated that the intervention led to a decrease in their intolerance of uncertainty, a psychological phenomenon that is implicated in anxiety and related to one’s ability to productively and functionally appraise unknown situations. Responses included: “Helped with decreasing attachment to things I can’t control”, “Tend to have a greater sense of agency with challenging life situations”, “… using STOP and RAIN to pause, step back, identify, make space has definitely help me to cope better with uncertainty”. Many participants commented on the benefits of the practical elements of the workshop and the subsequent increase in their therapeutic skillset, including the STOP and RAIN mnemonics (tools to remember how to navigate stressful situations).\n\nParticipants of this study indicated that having a dedicated space for self-reflection, particularly among a safe and supportive group, encouraged them to engage in more self-love which related to a theme of self-compassion. Responses included: “Given myself permission for self-care … ”, “More loving kindness to myself …”, “Being more loving/accepting of myself”.\n\nSome participants seemed able to translate the increase in self-care into motivation to improve their regular routine. Responses included: “It has rekindled my interest in mindful practice. Not only doing the homework but doing reading”, “It has brought me back to regular practice which I’m thankful for”, “Awareness of the importance of committing to a regular daily practice.”, “Accountability for practice = more structured practice”, “Practice self-compassion more frequently”.\n\nResponses included: “Using “STOP’ fairly often”, “Gave me ‘tool’ to use with my child. Re-affirmed some positive skills I use/have already with ‘data’ (i.e., name emotion, loving kindness, touch.)“, “Usefulness of STOP and RAIN – practical techniques … using STOP and RAIN to pause, step back, identify, make space has definitely help me to cope better …”, “Gave me skills for relaxation when I am stressed.”, “I particularly enjoyed the “what brings you joy” exercise and found myself asking my loved ones that question”.\n\nFinally, participants frequently discussed the positive impact that the above factors had on their interpersonal relationships: Responses included: “It has improved my relationships with my friends and family. I am less emotionally reactive with my spouse and children”, “I’m more present in my relationships”, “… more loving … of others”.\n\nMany participants indicated that they believe they became more effective in their interactions with clients, through a change in their capacity to manage their own emotions and distress tolerance, indicating an increase in mindfulness when working with patients: Responses included: “Easier to ‘stay’ with difficult patient and situations”, “I find myself more present with clients and less reactionary … to what clients are saying and feeling”, “I’m more aware of my ego slipping into the room and the habit of thinking of responses to clients before they are even finishing talking”, “Listening more and being quiet more”.\n\nAs well as through changes in their capacity to cope with workload and related stressors; responses relating to this theme included: “More loving kindness to myself as expressed by offering myself more time between sessions …”, “Learned skills to relax when stressed and this has enabled me to do more i.e., to be more efficient in finishing my tasks”, “Helped with work productivity and ability to slow down and cope with workload more effectively”, “I have a tendency to jump to problem solving/change strategies when not mindful – using STOP has helped me STOP, validate, then work toward change”.\n\nResponses also indicated that participants are likely to utilise the knowledge from the intervention when providing psychoeducation to clients: “Using the breath to help with present moment connection to my clients”, “I loved reflecting daily on some of the slogans … using these within my work”, “Helping clients connect more to their breath/sensations in their body”, “encouraging it more with patients”, “Realisation of the importance of sharing this practice with my client”, “I have shared the STOP technique with pain clients and children and their families and they inform of finding it helpful.”\n\nLastly, participants report a noteworthy change to the general relationship they have to their workplace as well as improvement to their workplace relationships: Responses relating to this theme included: “Less manifested irritation at other co-workers”, “It has provided an opportunity to engage with colleagues in a different way and a greater sense of community (in a small way) which could grow …”, “I have been taking more initiative at work … I feel more connected to my colleagues”\n\nOverall, the major themes of this study, personal and clinical benefits of mindfulness, can be conceptualised in the context of improvement in two key interpersonal domains. The first is emotional reactivity. Several participants reported a marked reduction in emotional reactivity, which tended to be described as a sense of being able to better notice their own emotional reactions and reflect upon them without immediately responding. This ability to “stop and reflect” was frequently associated with the respondent’s perceived improvement in their ability to listen and communicate. The second is listening and communication. Several participants described that they felt able to listen more deeply to clients during clinical work when emotionally distressing topics were being explored. Finally, a large proportion of participants described that they were able to communicate more effectively with family members and professional colleagues.\n\nSeveral participants discussed how the reduction in emotional reactivity allowed them more time to reflect on responses, rather than reacting to the emotion of the situation. Subsequent changes in listening and communication were often directly related to the reduced emotional reactivity. As such, it appears that when clinicians are able to advance their mastery over their own emotional processes, they are able to more effectively attend to other’s needs. This process is supported by the commonly used models of transference/counter-transference as well as mindful practice theory.44,45 Likewise, the combination of the above factors seems to have a commonly shared improvement on personal wellbeing through self-care, self-love, and increased connectedness to others.\n\n\nDiscussion\n\nThis study showed that a short, validated mindfulness course enhances components of, and overall mindfulness in mental-health clinicians, including psychiatrists. Critically, this finding was supported in the participants’ subjective reporting of their experience, which provided more nuanced insight into how the benefits might manifest themselves, primarily through the emotional, communication, and relationship benefits of the program. This study provides a novel synthesis of quantitative and qualitative data relating to the impact of mindfulness training in the sample.\n\nThe findings from this study validate the results of previous research, indicating mindfulness is protective against burnout, and provides valuable insight into possible contributing factors.46 Participants reported that after the intervention they were able to listen to clients more deeply and communicate with patients and family more effectively, themes directly related to the observation and describing FFMQ subscales. Both observing and describing have been positively correlated with active listening and empathy, which in addition to the clinical and therapeutic benefits, are likely to be protective against burnout.47,48\n\nNon-reactivity contributes to emotional resilience by way of allowing one’s emotions to occur without getting carried away.49 An increase in the non-reacting FFMQ subscale post MAPs was matched by participants’ narrative reports of reduced emotional reactivity. Emotional reactivity is directly correlated with burnout.50 Thus, moderating this with mindfulness techniques provides a direct mechanism of burnout prevention.\n\nThere are a number of limitations to this study. Self-selection bias may have selected for those clinicians more ‘psychologically minded’. Inherent limitations did not allow for blinding or control groups. Controlling for confounding variables such as the effects of medication or changes to the participants usual psychological treatment or social circumstances may have impacted the results. Furthermore, while the small sample size sufficed for the purpose of the pilot program, it will have almost certainly impacted on the statistical significance. High pre-test mean scores could have resulted in a ceiling effect, including awareness. Whilst no change in the mean PSS scores was observed, this could be explained by increased clinical demands and stressors in October, November, and December which is typically a busy time of the year for professionals in this field. Further studies can explore this through implementing numerous trials across the year.\n\nDespite the limitations, this study suggests personal and occupational benefits of mindfulness training in mental-health workers through enhanced resilience and ultimately prevention of burnout in mental health clinicians. To the best of the authors’ knowledge, this is the first mixed-methods examination of a mindfulness intervention in the sample population. The subject would benefit from further research using a larger sample size, which would also provide valuable information to support the scalability and practicality of such an intervention.\n\n\nData availability\n\nFigshare: 2016 Mindfulness Stats Complete.xlsx. https://doi.org/10.6084/m9.figshare.16566135.v1.51\n\nThis project contains the following underlying data:\n\n- Amherst 2016 Mindfulness Stats Complete.xlsx: Participant demographic information\n\n- PrePostPostRData_FFMQxlsx.xlsx: Pre- and post-test Five Facet Mindfulness Questionnaire scores\n\n- PrePostRawDataPSS.xlsx: Pre- and post-test Perceived Stress Scale scores\n\nFigshare: De-identified qualitative responses. https://doi.org/10.6084/m9.figshare.16757029.v1.52\n\nThis project contains the following underlying data:\n\n- Qual responses.pdf: Written responses to the two qualitative questions\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors wish to acknowledge Beth Wallace for delivering the Mindfulness Awareness Practices (MAPs) program.\n\n\nReferences\n\nMaslach C, Jackson SE, Leiter MP, et al.: Maslach burnout inventory. 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Publisher Full Text\n\nAlmalki S: Integrating Quantitative and Qualitative Data in Mixed Methods Research--Challenges and Benefits. J. Educ. Learn. 2016; 5(3): 288–96. Publisher Full Text\n\nFrancis JJ, Johnston M, Robertson C, et al.: What is an adequate sample size? Operationalising data saturation for theory-based interview studies. Psychol. Health. 2010; 25(10): 1229–45. PubMed Abstract \| Publisher Full Text\n\nWeiss RS: Learning from strangers: The art and method of qualitative interview studies. Simon and Schuster;1995.\n\nBlack DS, O’Reilly GA, Olmstead R, et al.: Mindfulness meditation and improvement in sleep quality and daytime impairment among older adults with sleep disturbances: a randomized clinical trial. JAMA Intern. Med. 2015; 175(4): 494–501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBower JE, Crosswell AD, Stanton AL, et al.: Mindfulness meditation for younger breast cancer survivors: a randomized controlled trial. Cancer. 2015; 121(8): 1231–40. 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Publisher Full Text" }	[ { "id": "100017", "date": "22 Nov 2021", "name": "Dr. Richard Mottershead", "expertise": [ "Reviewer Expertise I am a qualified researcher", "mental health practitioner with experience of supporting health and social care practitioners mindfulness and mental well-being." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the opportunity to review this research article paper. I have read the article with interest and have the following comments to make:\nThis is a well-composed paper, which will be of interest to all healthcare practitioners working within health and social care settings. The authors are embedded within the practice and seek to make long-standing contributions to the research literature and clinical practice for health and social care professionals through the advent of mindfulness techniques which consequently improve clinical effectiveness. The paper proposes a holistic benefit of enhanced mindfulness from engagement with service users to family members. The necessity for health and social care works need to be within the moment, not only within work but in family life which necessitates positive mental health and well-being in all aspects of life.\nIt is an approach to care that advocates self-love and self-care which are often secondary outcomes for health and social care professionals. The authors identify the challenges of generalising results and snowballing techniques that may lead to attracting participants willing and able to engage and adopt highlighted practices but notwithstanding. The research is an engaging insight to adopting evidence-based practices to improve service delivery as well as staff mental well-being.\n\nThe paper will undoubtedly be followed by others and I wish the authors well. Their work seeks to establish long-term benefits to the health and well-being of practitioners that deserve support in maintaining and enhancing resilience which will of course benefit service user care and recovery.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "101856", "date": "18 Jan 2022", "name": "Veena Singaram", "expertise": [ "Reviewer Expertise Health Professions Education" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors pilot a mindfulness program (MAPs) with a group of health practitioners, making this study a highly topical and useful study since this group of practitioners (and others) has been found to undergo symptoms of burnout in healthcare settings. The authors found that the MAPs program had positive benefits for their participants, both personally and professionally.\nThe study may be outdated due to the outdated references and the absence of current findings. Recent findings on mindfulness and healthcare professional wellbeing literature have proliferated the field of healthcare professional wellbeing especially since the advent of COVID-19, causing the authors to miss out on current developments in the field of healthcare worker wellbeing and burnout. This absence is understandable since the data were collected before COVID-19 began. Perhaps a word or two from the authors to signal and explain what may appear anachronistic to some readers.\nThe authors also comment on the participants’ high pre-test mean scores on the FFMQ and one wonders whether this may have been due to participants’ previous experiences with mindfulness-based programs and whether this does not partly explain the ceiling effect the authors speak about in their limitations.\n\nThough the study design was apt, the literature review and discussion spoke of burnout, yet the assessment tested perceived stress scales (PSS). Burnout is not the same as stress, thus I would suggest explaining why PSS was used instead of a burnout inventory assessing the different components as described in the first line of the literature review. Or, I would suggest changing the literature review and conclusions commenting on stress levels instead of burnout.\n\nIn terms of details of qualitative analysis, the steps were not described enough to make it replicable. Please add a description of how exactly the data was collated and analysed.\n\nOtherwise, the study is well put together, well written, and brings important attention to a group of healthcare practitioners professionally mandated to the mental well-being of others.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7753", "date": "31 Jan 2022", "name": "Ashwin Varghese", "role": "Author Response", "response": "We would like to thank the reviewers for their valuable input. We have addressed each comment in the revised submission. Specifically, we have expanded on the correlation between perceived stress and burnout, provided evidence to support this relationship, and explained why the PSS was used. We have provided a more thorough explanation of data collection and analysis. We have addressed the potential impacts of COVID-19 on the findings and made comments on the possible limitation of not knowing participants' previous experience with mindfulness." } ] } ]	1	https://f1000research.com/articles/10-1085
https://f1000research.com/articles/10-838/v1	20 Aug 21	{ "type": "Software Tool Article", "title": "TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data", "authors": [ "Francesco Reina", "John M.A. Wigg", "Mariia Dmitrieva", "Joël Lefebvre", "Jens Rittscher", "Christian Eggeling", "John M.A. Wigg", "Mariia Dmitrieva", "Joël Lefebvre", "Jens Rittscher" ], "abstract": "Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox – 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience.", "keywords": [ "Single Particle Tracking", "Diffusion", "Data analysis", "Python", "Graphical User Interface", "Simulation", "Microscopy" ], "content": "Introduction\n\nSingle particle tracking (SPT) is one of the most direct and employed methods to quantify particle dynamics in a sample using optical microscopy. As the name suggests, this approach relies on the identification and tracking of single particles in a sample, followed by the analysis of the detected trajectories. The analysis of particle trajectories is usually carried out using the description given by Einstein and Smoluchowski of Brownian motion1,2 to derive the diffusion coefficient of the objects. Given its nature as a data analysis method rather than a technique onto itself, SPT is indifferent to how the particle is detected, as long as the signal-to-noise levels are sufficient to identify it against the background. As a consequence of this, it is almost always necessary to label the sample with adequate target specificity, and it is important that the labelling should be sparse enough to enable single molecule level of detail, particularly when employing conventional, diffraction-limited microscopy.3\n\nAmong the earliest example of SPT experiments, we can mention the testing of Einstein’s theory of Brownian motion,4 during which tracking was executed by purely analog means,i.e. using pen and paper. The introduction of electronic digital camera detection and innovative microscopy systems have made it possible to extend the range of applications of SPT, which today reaches framerates up to 50kHz.5 More importantly, the use of large scattering tags is nowadays not necessary anymore to reach nanometer levels of localisation precision.6,7 The rise of such SPT-capable techniques, characterized by fast sampling and high data throughput, thus generates the need to adapt previously developed tracking and analysis pipelines.\n\nConventionally, trajectory analysis in diffusive systems is performed by monitoring the Mean Squared Displacement of the particle against the time increments, and analyzing them through the Brownian motion1 and similar models of diffusion.8,9 While this kind of pipeline has the advantage of being straightforward and easy to implement, it is not immune from potential pitfalls. As we elaborate further in the Methods section, the localisation error is a critical factor that is often overlooked.10 This aspect has become especially critical in recent times, given the rise in sampling rates accessible with modern hardware. Motion blurring, which is inherent in SPT given the movement of the target particles, is also often neglected as a potential source of measurement error.11 Overlooking these sources of error leads to overestimation of the detected diffusion coefficients,12 or to erroneously detected subdiffusion.13 These spurious deviation arise particularly at short time ranges, given that the particle displacements at these time frames is comparable to the localisation noise, and is thus particularly dangerous since many analysis pipelines often rely on the very first data points to extract relevant diffusion parameters.14\n\nSeveral toolboxes and algorithms have been produced to fulfill the need to identify and track single particles,15,16 and analyse their motion employing conventional mean squared displacement analysis,17 deep learning18,19 and other methods.20,21 However, while in many cases the source code is freely available, they are not platform-agnostic and require licensed software to be executed. On the other hand, tools available on open-source platforms, such as FIJI22 or ICY,23 are often more focused on the particle tracking itself, rather than the data analysis.\n\nThe TRAIT2D (Tracking Analysis Toolkit - 2D version), hereby presented, aims to supply the scientific community with an accessible, open-source, and platform-agnostic tool for particle tracking, simulation and analysis in two dimensions. In TRAIT2D, intuitive graphical user interfaces facilitate users without coding background to promptly access the tools. The tracking tool provided is the most specialised part of the package, as it was developed for particle tracking techniques with a high sampling rate, capable of acquiring long, uninterrupted trajectories, such as Interferometric Scattering (iSCAT).24 Thereby, the trajectory linking is based on the algorithm that favour strong spatial and temporal connections between consecutive frames. The analysis and simulation tools have a wider scope of applications. Once the particle trajectories are obtained, either via the provided tracking tool, or imported from other sources, two avenues are available to perform single particle trajectory analysis. The first and more widely adopted, is through the Mean Squared Displacement, which many users will find more familiar and is still conventionally employed. The second avenue, which we named Apparent Diffusion Coefficient analysis, provides more insights into the localisation error and a more intuitive way of monitoring the diffusion behaviour at different time scales. A statistics-driven approach allows the user to rely on objective parameters which diffusion model is more appropriate to describe the particle trajectories. The source code allows more advanced users to customize data analysis, allowing, for example, the introduction of user-defined diffusion models that integrate seamlessly with the proposed analysis pipeline. Another useful tool we introduce is the track simulator. This allows the user to generate particle trajectories on a homogeneous plane, or on a surface divided in compartments where the particle can be transiently confined, with a high degree of customisation. We also added the possibility of generating movies from the simulated trajectories with variable levels of signal-to-noise ratio, and a user-specified Point Spread Function. This last module can serve as a validation tool of further particle tracking and analysis pipelines.\n\n\nMethods\n\nThe particle tracking is developed to support a quantitative analysis module which requires extracted trajectories. We introduce a tool which allows users to adjust tracking parameters, preview results and save final trajectories.\n\nThe pre-processing of the image sequence is optional and can be applied prior to the tracking algorithm. The tracking is implemented as a two-step process. Firstly, the particles are detected using spot enhancing filter (SEF)25 and sub-pixel localisation is estimated by the radial symmetry centre approach.26 Secondly, the detected particles are linked based on their spatial and temporal locations.\n\nThe current tool was developed for the iSCAT imaging technique and the pre-processing step is built in accordance to the technique requirement. To distinguish the molecules of interest from the background, the image sequence is divided by a flat field. The flat field represents an undesired static background scattering, and it is calculated by the pixel-wise temporal median filter over a set of frames (1000 frames in the current version). The background is calculated as a temporal average of the entire image sequence and subtracted from each frame. At last, the movie is normalized and brightness adjustment is applied to the image sequence.27 Although the pre-processing step is developed for the iSCAT it can also be employed for other techniques as a background subtraction tool.\n\nThe particle detection exploits a spot enhancing filter (SEF) to enhance the particles and reduce correlated noise in the image. The SEF can be described by the convolution of the original image with a Laplacian-of-Gaussian (LoG) kernel followed by global thresholding to extract the spots.\n\nwhere σ is a standard deviation of the LoG. The threshold is defined by the average intensity of the image and its standard deviation, weighted by a constant c. This constant together with LoG parameter σ can be defined by the user. The local maximum of the image identifies a set of the detected particles in each frame. Further refinement of the particle coordinates (sub-pixel localisation) is implemented with the radial symmetry centre approach.26 It provides a faster execution time due to its non-iterative nature, while achieving high accuracy. The size of the region of interest for the sub-pixel localisation and the limitation for the detected peak size can be set manually.\n\nThe iSCAT imaging technique provides high temporal resolution. The small displacement of the particle between neighbouring frames allows reducing complexity of the linking algorithm and increase the computational speed. The linking approach focuses on strong spatial connection between frames. Hungarian combinatorial optimisation algorithm28 is employed for the task of data association. The tolerance of the algorithm towards the spatial and temporal distance can be set by the parameters in the graphical-user interface. The assembled tracks can contain gaps in temporal domain due to the failed detection in one or few frames. These gaps are filled taking into account detections in the neighbouring frames and sub-pixel localisation. At the final stage, all the trajectories are filtered based on their length.\n\nIn this and the following section, we will briefly elucidate the analysis methods implemented in the TRAIT2D software package. The first method, which we refer to in the code as ”MSD analysis“, consists of the approach to determine the physical parameters of diffusion dynamics based on the behaviour of the mean squared displacements against the corresponding time interval.\n\nIn the case of MSD analysis, the MSD is initially calculated according to the definition:\n\nwhereby tn = nt0 indicates the n-th time point from the initial time t0, and r_(tn) indicates the two-dimensional position vector at time tn. Notice that this notation, as well as the entire analysis pipeline hereby implemented, supposes that the time interval between localisation is constant. According to Einstein’s theory of Brownian Motion generalized for higher dimensionality,1,4 this quantity is linked to the diffusion coefficient, D, and time by the formula:\n\nin which n indicates the dimensionality of the system. In the rest of the section, we will assume d = 2 to simplify the formalism. In order to account for experimental finite localisation precision, it is necessary to introduce an additive term to 329:\n\nThe above formulation assumes that the localisation error in the x and y directions is identical. This formula has been generalized to include the possibility of anomalous subdiffusion30:\n\nWhile the user can, of course, fix the localisation error value according to their chosen metric, it is also possible to leave it as a floating parameter. This is theoretically preferable from the point of view that the detection of moving particles is by necessity less precise than that of immobile particles, which are often used as a reference to estimate the localisation precision of a microscopy system. The effect of motion blurring, to which this loss in precision is ascribable, is represented by a negative term by the form11:\n\nwhere R is a term which depends on the illumination mode of the chosen microscopy technique, and obeys 0 ≤ R ≤ 1/4. It is especially interesting to notice how this term is dependent not on the time variable, but rather to the time resolution of the measurement. As a consequence of this, non-blur-corrected MSD data may suffer from considerable distortion at short time intervals, which coincidentally are the ones that are considered most important in determining the kind of motion the particle undergoes.12,31 When this term is added to Eq. 4 and Eq. 5, it gives rise to the formulas:\n\nand\n\nwhich are ultimately used to perform curve fitting and extract the relevant parameters.\n\nThe apparent diffusion coefficient (ADC) analysis differs from the MSD analysis mainly for the central role given to the diffusion coefficient compared to mean squared displacement. We define the ADC as:\n\nthereby enabling us to switch to a diffusion coefficient-centric representation. The nomenclature ”apparent” is adopted to stress how this quantity is still affected by the localisation error and not the absolute value of the diffusion coefficient. Dividing both terms of Eq. 7 by 4tn1−2Rn, and with some elementary algebra, it is possible to derive:\n\nBy substituting in place of D(tn) the appropriate expression for the diffusion coefficient for a specific model, it is therefore possible to obtain a complete model to fit to the apparent diffusion coefficient data, obtained according to Eq. 9. In principle, it is possible to leave the localisation error δx2+y22 as a free-floating parameter in the fitting operation, to better estimate the localisation error for moving particles.\n\nThe software package hereby presented comes with three built-in diffusion models,12,32 corresponding to free (Brownian) diffusion\n\nconfined diffusion\n\nand compartmentalized diffusion, which is the combination of the above two models\n\nThe nomenclature for these models follows the convention that DM is a constant diffusion coefficient, corresponding to theoretical Brownian diffusion, and that τ is the characteristic residence time in the confinement zones described by the confined diffusion model. Dμ is the microscopic diffusion coefficient observed in the aforementioned confinement zones, which can also be expressed as\n\nwhere L is the average confinement zone size.9 It is apparent that while this data analysis pipeline appears more complex, it however readily provides an efficient way to directly extract relevant physical parameters from the particle trajectories. We point out that, although the code we hereby describe comes with the aforementioned built-in models, chosen according to the authors specific research interests, a documented and simple procedure is in place to define a new model, according to the end user-specific wishes. The only restriction is that the custom-defined models can only be used when the analysis module is imported in a Python Script. However, the analysis pipeline will integrate this addition seamlessly alongside the other models, or run exclusively on the user-defined models.\n\nThe selection of the best model to describe any given track is done by statistical means. Each of the models is fitted to the Dapp data, and the Bayesian information criterion (BIC) for each model is calculated according to the formula33:\n\nin which n is the number of data points the model is fitted to, the RSS is the residual sum of squares, and k is the number of degrees of freedom, i.e. the free parameters, of the fit. The most adequate model to describe the diffusion motion is then the one with the lowest value of BIC. Since the BIC is not a goodness-of-fit metric, the analysis function will also perform a one-sided Kolmogorov–Smirnov test, comparing the original ADC data against the results of the fit.\n\nIn order to validate the analysis methods described above, we have developed a simple simulation framework. Our tool can create synthetic particle tracks using various diffusion models. Furthermore, the tracks can be converted into synthetic movies to test the particle detection method.\n\nThe free (Brownian) diffusion was simulated by drawing a random walk on a two-dimensional virtual square space of side L, entered by the user. The particle is initialized in the centre of the square. The user also has the ability to select the value for the diffusion coefficient D and the time step dt. The position of the particle is updated at each successive time step in the x and y direction separately:\n\nThis set of equations does not strictly respect the condition that (x(t + dt)−x(t))2 + (y(t + dt)−y(t))2, since doing so would not account for the variability that happens in real simulations. Instead, the theoretical displacement D*dt is multiplied by a random number (“rand“) extracted from a normal distribution of mean μ = 0 and σ = 1.\n\nThe hop diffusion motion was simulated according to the code used for,34 kindly provided by Dr. J. Keller–Findeisen as a Matlab script, and transcribed into Python by the authors. First of all, a virtual surface of arbitrary dimension is split into an arbitrary number of compartments as a Voronoi diagram with random seed. To each of the compartments, a unique identifier is assigned. A particle is then generated in the centre of the plane, and its motion is simulated in the same way as the Brownian diffusion case. However, an additional condition is in place to simulate the hopping motion. A probability is assigned to the particle of jumping between one compartment and the other, called hopping probability (HP). Every time that the randomly generated displacement would move the particle to a different compartment, a random number between 0 and 1 is extracted. Should this number be less than the value of HP initially fixed, the displacement calculation is repeated in order to keep the particle in the same original compartment.\n\nConfined diffusion can easily be simulated by fixing HP = 0.\n\nWe have created a simple movie simulator to obtain a synthetic movie from a list of detected or a simulated track. This simulator is inspired by a similar noisy holographic image simulator.35 Apart from a mandatory list of tracks to simulate, otherDM, parameters required for the simulation are the spatial (rxy) and temporal (rt) resolutions, the signal-to-noise ratio (SNR), the background signal level (μbg), and the Gaussian noise level (σbg2). Optional inputs are a point-spread function (PSF) stack, which can either be obtained experimentally or estimated with a model of the microscope, such as with DeconvolutionLab2.36\n\nThe simulator is initialised using the given tracks. The number of time frames, the minimum, and the maximum spot positions in X and Y are extracted from all tracks. These are used to initialise the simulation grid. The (xmin, ymin) and (xmax, ymax) positions define the simulation grid width and height. If the grid needs to be square, the min and max between (xmin, ymin) and (xmax, ymax) are computed respectively to define the grid shape. The simulation grid is discretized using the spatial resolution rxy. For example, a track whose minimum position is (0,0), and maximum position is (10,10), and for a simulation resolution of rxy = 1, will have a shape of 11 × 11, where each grid position represents a pixel of size 1 × 1 μm2. Furthermore, if the maximum time frame contained in a tract is 10, and if the temporal resolution is set to rt = 1, the simulated movie will be of shape (11 × 11 × 11), where the first two dimensions represent the spatial dimension, and the last is the temporal dimension.\n\nOnce the simulation grid is initialised, we first add a background signal B(x,y,t)∼N(μbg,σbg2) everywhere. Each pixel background intensity follows a normal distribution of mean μbg and of variance σbg2, and each simulated background pixels are independent and identically distributed (i.i.d.) random variables. In other words, at this stage there is no spatial or temporal correlation between the pixels intensities. We are using the util.random_noise method from the scikit-image Python module.37\n\nNext, for each spot position in each track, we discretize the position in both the spatial (x,y)→(mx,my) and temporal dimensions (t)→(mt) using\n\nwhere i ∈{x,y,t} represents the x, y, and t dimension of the spot positions respectively, xi is its position as recorded in the tract, xi,min is the minimum position within the tract, ri is the simulation resolution along the ith axis, and [⋅] represents the roundingDM, operation to obtain discrete positions mi within the simulation grid coordinate framework. Each spot position is added iteratively to the simulated movie, and its intensity is given by the desired SNR for this simulation. At this stage, the simulated movie can be described by the equation\n\nwhere δ(x − mi) is a n-dimensional Dirac delta function, x = (x,y,t) is a (3,1) vector representing the spatiotemporal position within the simulation grid, mi = (mx,i,my,i,mt,i) is the discretized vector position within the simulation grid of the ith spot, and N is the total number of spots among all tracks to simulate. If a PSF is given as input, it will be applied at this stage with a convolution operator. If a 3D PSF stack is given, the central slice of the stack (corresponding to the focus position) will be used instead of the whole 3D stack for the simulation. The PSF can either be an experimentally acquired stack or a simulated volume, for example using the DeconvolutationLab2 with the microscope’s configuration.36 To consider the boundary effects, we use zero padding. Thus, the simulated volume is\n\nwhere ⊗ is the convolution product. Finally, i.i.d. Poisson noise is simulated for each pixel separately, using the value of M(x) as the Poisson distribution parameter. The Poisson noise is simulated using the python module random_noise from scikit-image. Thus we obtain\n\nwhere\n\nThe simulated movie is exported as a tiff stack or an avi file. Figure 3 is a static example of a simulated movie frame using a single simulated track. The image on the left is a movie frame before the PSF convolution and the Poisson noise simulation, and the image on the right is the final simulated movie frame after these operations.\n\nTRAIT2D is implemented entirely in Python using available packages.\n\nThe software components (simulation, tracking, analysis) have been split into separate modules that have graphical user interfaces (GUIs) available or, in the case of the simulation and analysis libraries, can be imported in Python scripts.\n\nTRAIT2D uses an object-oriented approach for its workflows:\n\nThe simulation library uses Diffusion objects which hold the parameters and mathematical representation of a diffusion model. Specific models inherit from the base Diffusion class. Simulation results are stored to the simulator as a Python dictionary.\n\nIn the analysis library, tracks are represented as Track objects, which can be created from such a dictionary, a Pandas data frame or a CSV file. Multiple tracks can be batched in a ListOfTracks object. Analysis is executed by calling the corresponding methods on these objects and results are stored to them.\n\nA unique property of the analysis library is the ability to define and add new diffusion models DM,through a “singleton” class called ModelDB. This functionality is only exposed when importing the analysis module from a Python script but not from the GUI.\n\nTRAIT2D requires at least a Python 3 (recommended ≥ 3.7) to run. The software also depends on additional packages: Graphical user interfaces are provided through PyQt (analysis) and Tkinter (simulation, tracking). Scipy, matplotlib and pandas are used for model fitting, plotting and loading data, respectively. These and other dependencies are automatically installed when installing from source using the supplied setup.py file or from the Python Package Index (PyPI).\n\n\nUse cases\n\nThis section outlines basic use cases for TRAIT2D. The complete documentation as well as a list of tutorials as available at https://eggeling-lab-microscope-software.github.io/TRAIT2D/.\n\nThe central component of the TRAIT2D software package is the analysis module, through which it is possible to extract physical parameters from the particle trajectories. There are three possible avenues for the user to analyse their data (Figure 1). By using the tracker module, the user can import their timelapses, as TIFF stacks, into the GUI and extract particle trajectories, which can then be exported as a formatted CSV file, ready to be imported in the analysis GUI. Alternatively, if the trajectories are already available to the user through a different particle tracking tool, they can be imported as well, on condition that their formatting is compliant with the format required by the analysis module. Finally, the user could also generate a control dataset through the simulator module, either through the GUI or as a script. The simulated trajectories will then be exported as a properly formatted CSV file, or a compliant data structure in the case of the user script. The Analysis GUI will perform the data analysis following either the MSD or the ADC pipelines (see Methods section), as indicated by the user. We stress that while the GUI exclusively uses the predefined set of models (see Eq. 11, 12 and 13) for the analysis, the module itself allows the addition of more diffusion models as well as batch processing when used in custom scripts, adding an ulterior degree of flexibility.\n\nSchematic of the workflow of the TRAIT2D software package, highlighting the three possible paths to feed data to the analysis module: (left) through the tracker, starting from a TIFF stack of the diffusing particles, center importing trajectories otherwise obtained as a CSV file, (right) or by simulating particle trajectories with the simulator module, starting from user-defined parameters.\n\nThe analysis library and GUI expects track data to be supplied in a specific format although column names and units are adjustable. At the time of the first release, the trajectories can only be imported from CSV files. A track file must include at least three columns, which contain the localisation time t as well as the coordinates x and y. The order in which these columns appear is not important. A unique numerical identifier id must be added as a fourth column in files which contain multiple tracks. This identifier will match each localisation with a specific track.\n\nWhen working with the analysis library as an imported module inside a Python script, tracks can also be directly loaded from a dictionary or a Pandas data frame as long as they contain the keys described above.\n\nThe package containing the full software can be downloaded directly from PyPI, using the command:\n\n\n\nAlternatively, the source code is available at the Github repository:\n\nhttps://github.com/Eggeling-Lab-Microscope-Software/TRAIT2D.\n\nOnce installed, the different GUIs can be launched by entering trait2d_analysis_gui, trait2d_simulator_gui, or trait2d_tracker_gui in a Python-enabled environment.\n\nThe analysis and simulation libraries can be used as modules in Python scripts by importing either trait2d.analysis or trait2d.simulators. This exposes additional features such as bulk analysis and adding custom diffusion models and allows the user to program pipelines for their specific use case. The tracker software is only available as a GUI. 22\n\nThe particle tracker is available only as a GUI tool at the time of writing. It can be accessed by typing the command trait2d_tracker_gui in a terminal with a compatible Python installation. The application accepts 8 bit TIFF stacks as an input. It is possible to run a simple pre-processing step with background subtraction. The tracker GUI provides options to set parameters, and to preview particle detection results frame by frame. Once the parameters for detection and trajectory linking are deemed satisfactory by the user, the application will perform the trajectory linking over the image stack, and export another tiff stack where the detected trajectories are superimposed as a coloured track over the original data (Figure 2). The user can thereafter choose to export the particle tracks as a csv file, with formatting compatible to the analysis tool.\n\n(a) Example of the output TIFF file, exported by the program after the tracking procedure has been completed. The tracker has found three tracks: one over the full movie length (blue) and a shorter track confined to the sport in the bottom-left corner (yellow). The latter is cut off due to the spot being close to the border. (b) Graphical result of the analysis, through the ADC pipeline, of the track in blue, using the three built-in models, with an opportune analysis interval, initial values and bounds for the parameters.\n\nWhile the majority of the use cases presented here will be focusing on the more elaborate ADC analysis pipeline, a quick overview of the MSD analysis pipeline will be given in this section as well.\n\nMSD analysis uses only the built-in linear (Eq. 7) and power (Eq. 8) models for fitting, and there is no option to add custom models.\n\nOther features, such as bulk processing, however, are also available for the MSD pipeline and work similar to their ADC counterparts.\n\nBelow is an example illustrating the process of importing a single track from a file and running MSD analysis on it:\n\n\n\nIn order to be analysed, a track must be contained in a compatible file or data structure (see above). Data analysis can then be performed through either the analysis GUI, accessible by typing trait2d_analysis_gui in a terminal with a compatible installation of Python, or through a user script by importing the trait2d.analysis module. For the use of the analysis GUI, we would reference the documentation https://eggeling-lab-microscope-software.github.io/TRAIT2D/analysis_gui.html, where it is extensively described. The following code snippets exemplify how to load a single track from one of the example files provided with the code, and analyse it using a Python script.\n\nFirst of all, the track data needs to be imported as a trait2d.analysis.Track object:\n\n\n\nIn order to proceed with the analysis, the models need to be defined. The package comes with three default models (Eq. 11, 12 and 13), which can be handily registered in the trait2d.analysis.ModelDB object. All models added to this singleton object will then be used to analyse the track data. Lower and upper bounds, as well as initial values, can also be easily set on a per-model basis. The following code snippet shows, as an example, how to import the built-in brownian diffusion model (Eq. 11), and define the bounds and initial values necessary for the fitting operation:\n\n\n\nFor the other models, the procedure is identical, however more or less parameters should be initialized, as required by their expression. Once the ModelDB has been populated with the desired diffusion models, the analysis is performed via the command: DM,\n\nThe analysis module saves the analysis results to the trait2d.analysis.Track object, so they can easily be accessed at a later point. More options concerning the whole analysis pipeline are also available in the documentation. Several examples are also provided as downloadable Jupyter Notebooks.\n\nThe analysis module allows to define custom diffusion models which can be used in the analysis pipeline, and integrate seamlessly into it. This is done by defining a class which inherits from trait2d.analysis.models.ModelBase. This functionality is only available when importing the module in a Python script and not from the GUI.\n\n\n\nThe newly defined model can be added to trait2d.analysis.ModelDB, in addition to or instead of the built-in models and will be used in all subsequent analyses. This functionality is only available when importing the module in a Python script and not from the GUI.\n\nWhen using the analysis module inside a Python script, bulk processing of multiple tracks is available. For this, the trait2d.analysis.ListOfTracks class is used. In the following example, a CSV file containing multiple tracks is loaded. In addition to MSD and ADC analysis, ListOfTracks also exposes useful methods such as ListOfTracks.plot_trajectories or ListOfTracks.adc_summary to quickly visualise all contents and analysis results of the contained tracks. Bulk processing is also available for MSD analysis via ListOfTracks.msd_analysis.\n\n\n\nTrack simulation can be done either from the GUI, which can be accessed by typing trait2d_simulator_gui in a terminal with Python available, or by importing a simulator from trait2d.simulators.\n\nThe following code example shows the simulation of a track using the imported module and the built-in Brownian diffusion simulator.\n\n\n\nThe GUI also allows for the creation of an image sequence from the simulated track or a loaded trajectory file. Note that this is currently not possible from the imported module inside a Python script. Figure 3 shows such a simulated particle diffusion movie.\n\n(Left) Frame before and (Right) after the convolution by a PSF and the simulation of Poisson noise. An animated version of this simulation is available in this project Github repository.\n\n\nConclusion\n\nTRAIT2D is a Python-based, open source and easily deployable toolkit for the analysis and simulation of two-dimensional SPT data. The combination of particle tracking, simulation, and trajectory analysis in the same package provides a number of benefits for the user. Another beneficial feature of this software is its user-friendliness for inexperienced users, thanks to the graphical user interfaces provided for each module. Nevertheless, the potential for customisation is retained and the scope of the module can be readily expanded by users with more advanced coding background.\n\n\nData availability\n\nThe tracks used throughout the examples above are available in the source tree in the examples folder: track1.csv, track2.csv, and track3.csv each contain a single track in the format expected by TRAIT2D. three_tracks.csv contains the same files which are identified by a separate id column. These example files report the trajectories obtained by performing particle tracking on three distinct timelapses of 40nm diameter, streptavidin-coated gold nanoparticle linked to DSPE-PEG(2000)-biotin lipid analogues diffusing on an artificial membrane (Supported Lipid Bilayer) created on glass support.\n\n\nSoftware availability\n\n\n\n• Software available from https://github.com/Eggeling-Lab-Microscope-Software/TRAIT2D/releases/ and https://pypi.org/project/trait2d.\n\n• Source code available from https://github.com/Eggeling-Lab-Microscope-Software/TRAIT2D.\n\n• Archived source code at time of publication: https://doi.org/10.5281/zenodo.4725268.\n\n• License: GPL-3.0 License.", "appendix": "Acknowledgements\n\nFR and CE would like to acknowledge Dr. B. Christoffer Lagerholm for his invaluable scientific advice. The authors would like to thank Dr. J. 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PubMed Abstract \| Publisher Full Text Reference Source\n\nVan Der Walt S, Schönberger JL, Nunez-Iglesias J, et al.: Scikit-image: Image processing in python. PeerJ. 2014; 20140(1): 1–18. 21678359. Publisher Full Text" }	[ { "id": "93050", "date": "22 Sep 2021", "name": "Xavier Michalet", "expertise": [ "Reviewer Expertise Single-molecule biophysics", "single-photon detectors", "fluorescence spectroscopy and 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\nSummary:\nThis manuscript by Reina et al. describes a Python package implementing 1) single-particle tracking (SPT); 2) single-trajectory analysis (STA) by MSD (isotropic Brownian diffusion and isotropic anomalous diffusion) or ADC (Apparent Diffusion Coefficient, an acronym coined by the authors to designate a single-MSD point estimate of the diffusion coefficient); and 3) SPT movie simulation. The package is provided as a series of Python modules installed using the pip install command and can be run as 3 separate GUIs, as Python command lines, or within Jupyter Notebooks. The package comes with fairly detailed documentation (ReadTheDocs format, with code comments used to describe the different functions and classes in the module, and additional illustrated text description of the installation procedure and basic tutorials).\nThe manuscript consists of a very basic introduction on the basics of SPT and single-trajectory analysis and a brief overview of a few examples of how to use the 3 different modules (SPT, Analysis and Simulation), and provides the necessary links to the Github repository and documentation. It is fairly well written and is sufficient to assess what the package will allow an inexperienced user to do. Basic attempts by this reviewer to test the different GUIs, followed by some failures or lengthy lists of warnings, seem to indicate that minor additional details and guidance could be useful for potential inexperienced users. The samples of code checked by the reviewer appear well-documented, but an overview of the relationship between different functions and classes might be useful for developers interested in using this project as a basis for their own tools (contributions do not appear to be discussed in the Github repository).\nThe following are a few remarks which the authors could potentially use to remedy these deficiencies.\nIntroduction\nWhile this is not a review article, it seems that the introduction ignores a large body of literature and code on SPT and STA. For instance, the Particle Tracking Challenge paper (2014)1 could be cited as it was an interesting effort to compare the multitude of isolated tools existing at the time, and would have provided a convenient set of test files to run the present software package on. There are many (literally dozens of) freely available packages to track and analyze single-particle data (easily found by an internet search with single-particle tracking as the keyword) and not all are using Matlab or “require licensed software to be executed” (Matlab can usually be replaced by the open-source Octave package), some are web-based (Spot-On) and many come with GUIs; therefore, some of the arguments put forward by the authors to justify their effort are somewhat artificial. This does not minimize the appreciable effort to document and simplify access to this type of tool for the novice user, although some additions might be needed (see later, GUI tests).\nTechnically, the paper only scratches the surface of SPT and STA, but for obvious reasons since it is primarily a software presentation manuscript. However, since it appears, at least in part, targeted to novice SPT users, it would seem important to emphasize some important simplifications used in its algorithm implementations. For instance, the authors use the “Radial symmetry center approach” (which I was not aware of) because of its speed, but it is assuming radial symmetry, which might not always be appropriate (e.g. in the case of double-helix or astigmatic PSFs or simply long integration combined with fast diffusing particles). From a coding perspective, it could be useful to document how to replace this part of the code with another (or better yet, provide an example of such an optional alternative fitting algorithm – FluoBancroft comes to mind).\nWhen mentioning the blurring effect on diffusion analysis, the paper by Goulian & Simon is seminal, but in addition to Michalet 2010 already cited, Berglund 20102, Michalet & Berglund 20123 and Vestergaard 20124 went some extra length to show how this affected the uncertainty on estimated parameters when performing optimal analysis (a question addressed further below). The authors might find it useful to point their readers to these papers (or not: it is not my intention to be self-serving, only to point out the often forgotten and rediscovered fact that this is not a completely trivial matter), which are a good starting point to follow the more recent literature on parameter estimation in more complex models.\nTrajectory analysis\nMSD analysis, even with a large number of locations, is subtle (as just mentioned, see e.g. Fig. 7 of Michalet & Berglund 20123), in the sense that the number of MSD points used has an effect on the precision of the extracted parameters. In fact, there is an “optimal” number (or range of numbers) to be used if one wants to minimize the error on the estimated parameters, and to some extent, this number can be automatically determined. Is that number the “Range” parameter found at the bottom of the Single-Track Analysis GUI window, and in that case, could an optional determination be offered? How is the user supposed otherwise to guess the best value without any theoretical insights?\nAnomalous diffusion analysis in the presence of an error is treated only in an ad-hoc manner in Eq. (8) (I could not find mention of it in ref. [30]) and it is therefore not quite clear how accurate the determination of either error, D_alpha or exponent alpha, is (not mentioning the eluded optimal number of points to use in the analysis). The point is, as discussed extensively in the recent literature, that MSD is almost certainly not an optimal method to analyze anomalous diffusion trajectories. I will not comment on the ADC approach, but clearly, the same comments would apply (optimal number of points and uncertainty on extracted parameters). In this respect, the “error” outputs on fitted parameters provided by the STA module are a bit mysterious (it would be a simple addition to write down their definitions in an appendix).\nTracking\nI would likewise recommend discussing (or cite a reference for) the LoG kernel and what its role is in image analysis. There are two user parameters to specify when implementing this kernel and it is unclear how to best choose them. In fact, the same question arises for the radial symmetry center algorithm (2 parameters) and the connection algorithm (2 parameters).\nAll the previous discussions about tracking uncertainties, SNR, etc., seem for naught when contemplating the CSV file format for SPT trajectories, as it doesn’t include fields such as integrated intensity or localization uncertainty. Clearly, these parameters should be outputs of the SPT part and could/should be used to take into account as much possible information on the data as possible (for instance, to set the localization uncertainty to a fixed value?).\nSimulations\nA priori, the correct simulation of diffusion with coefficient D is to use displacements distributed as N(0,sqrt(2D dt)) (see e.g. SI of ref. 10 ). The authors use instead sqrt(D dt) N(0, 1), which appears different based on the linearity of normal distributions (aN(0,s) + b = N(b,\|a\|s)). Please clarify.\nOn P7, provide an explicit definition of the SNR, as there are several ways to define something that can play the role of SNR.\nTest of the provided code\nI only tested the GUIs after installation in a dedicated Anaconda environment. Unfortunately, I encountered a few issues, which the authors might be able to address in the documentation.\ntrait2d_analysis_gui: Tested on the provided trackn.csv files, the terminal window spits out a large number of warnings.\nHow is the user supposed to deal with those (and should they care)?\n\nWhat is the “Max. fit iterations” parameter for?\n\nWhat is the “Use parameters as initial guesses” checkbox for?\n\nWhat is the “Range” parameter for (it seems to be used to determine the number of data points (MSD or ADC) used for analysis)? I am assuming this is equivalent to the “fit_max_time” parameter discussed in the command line version (P 10) but again, this could be leaving a lot of freedom to a user unaware of the literature.\nTrait2d_simulator_gui: Tested with default parameters and “Free Diffusion” resulted in errors in the Terminal window (for both Generate and Show Track). Same with Hopping Diffusion. It would be useful if the default parameters actually resulted in usable data. Minor tinkering resulted in viable results, but the generation of a simulated movie can take a considerable amount of time (and doesn’t seem to be parallelized); therefore a progress bar or some indication of the current frame number could be helpful. An indication of the resulting movie size could also be useful beforehand (those files can grow rapidly large when using the wrong parameters).\nTrait2d_tracker_gui: It would be nice to provide an image stack example (and 8-bit TIFF stacks might be limiting in a world where most cameras output 16-bit images). I used the test image stack provided with TrackMate (a FIJI plugin) and the default parameters (with Light Spot option) but that failed (empty track). The file I generated by simulation using the previous module worked somewhat but it was difficult to figure out optimal parameters, and once tracking is started, it appears impossible to abort it (the progress was clearly indicated by the terminal output). Enabling partial initial processing of large stacks could prevent this kind of problem. Likewise, providing a short example stack – for instance, that discussed in the text – and initializing the GUI with appropriate parameters could help a user play around with parameters and understand the influence of each of the 7 important ones. Saving/loading analysis parameters for future reuse with similar datasets could also be useful.\nTypos:\n\n“otherDM”, “roundingDM”, “diffusion models DM”, “”command: DM” throughout the text.\n\nThe sentence “allows the user to rely on objective parameters which diffusion model is more appropriate” does not make sense.\n\nP4: elucidate >> discuss.\n\nP6: The sentence “the condition that (x(t + dt)-x(t))^2 + (y(t + dt)-y(t))^2,…\" does not make sense.\n\nP8: stored to the simulator >> stored in the simulator\n\ncan be batched >> can be pooled\n\nstored to them >> stored in them\n\nat least a Python 3 >> at least Python 3\n\nP10: compatible to the analysis tool >> compatible with the analysis tool\n\nReferences: some of the journal volume numbers have 3 or 4 digits, which appears to be a mistake.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "7608", "date": "31 Jan 2022", "name": "Francesco Reina", "role": "Author Response", "response": "We thank the reviewer for this extensive and constructive report. We acknowledged his comments, and have accordingly improved the manuscript, code, and documentation. Herein, we address all the reviewer’s concerns. Introduction We appreciate the time and effort spent by the reviewer to analyse the introductory paragraph. Given the extensive body of literature on SPT data analysis, and with the good intention of not expanding the introductory paragraph too much, indeed some literature has been glossed over. We have included the Particle Tracking Challenge paper among the references, for a more comprehensive overview of work already carried out on SPT and STA data analysis. On the topic of why the tool has been developed, the reviewer's comment has sparked a constructive discussion among the authors, which led to a reframing of the tool in the context of Single Particle Tracking accessibility and the tools already available. This now reflects more accurately the intentions of the authors, and we thank the reviewer for their honesty that allowed us to further consider this point. We have highlighted limitations of the radial symmetry centre method and the reason it was selected among other existing methods. We also have proposed alternative solutions, providing general direction to replace the current sub-pixel localisation function in the code. We have chosen to address the choices of algorithm implementations as pointed out by this reviewer. In particular, as mentioned by the reviewer, we specifically addressed the choice to adopt the Radial Symmetry Center approach in order to fit the PSF of the diffusing particle in the movie as a deliberate decision originated by the research environment and the kind of data that favoured the inception of the present tool. Nevertheless, together with a general invitation to contribute to the code, a roadmap on how to implement a different tracking algorithm is provided. The short but comprehensive list of works suggested by the reviewer has been taken into account, and an invitation to further reading on the appropriate amount of points to take into consideration for the analysis has been added as a precaution to the correct use of the tool. Trajectory Analysis Naturally, we concur with the reviewer that the number of MSD points, and indeed the model fitting methodology, have a significant effect on the precision of the parameters extracted, as this has been derived in several papers mentioned thus far in the reviewer report. The “Range” parameter, in the present tool, is a visual aid for the user to keep track of the time range considered in the model fitting. We acknowledge the fact that in the paper pointed out by the reviewer a rough way to estimate the optimal number of points is provided, which would prove useful in the analysis of Brownian diffusion especially, however, we deem this to be beyond the scope of this initial release. Nevertheless, given the open-source nature of the project, a contribution in this direction could also be implemented in the main code, and we rendered this process easier by adding specific guidelines for contribution (whose previous lack we thank the reviewer for pointing out). We, therefore, invite the reviewer to contribute an issue to our GitHub once the review process is complete so that the topic can be further debated and the tool developed in this direction. The inclusion of Ref [30] before equation (5) is an oversight by the authors and it has been amended, we apologize for the confusion. The inclusions of Eq. (5) and (8) are included as the MSD is still widely used as a measure to at least qualitatively evaluate anomalous diffusion [2], and thus its inclusion was deemed worthwhile. It should be noted, however, that while in the GUI, in the present release, both Eq. (7) and (8) are always fitted to the experimental data, this is not the case for a customized script including our library, which allows the user to freely exclude either, or define their own models altogether. Insofar as the accuracy of the determination of the parameters is concerned, we have added statements in the “Implementation” section detailing which algorithms in the SciPy library are employed and how the errors are thus calculated. Regarding the absolute accuracy of the method, we have emphasized the need for the user to further explore the topic, and we are sure that the present report, being publicly available, will also provide further guidance to novice SPT adopters. The comment on the ADC analysis regarding the errors of the estimated parameters was resolved by commenting on the model fitting procedure as for the MSD analysis. A reference to the range as intended by the reviewer is further added for the case of the free diffusion, in Eq. (11), since in this case it clearly would apply. Tracking The reference to the Laplacian-of-Gaussian and a short discussion of its role is included in the manuscript, as well as the influence of the LoG kernel and threshold parameters into the spot detection. Suggestions on the parameter settings are also provided in the software manual. We have added a link to the manual in the software interface to make it convenient for the user to find the information. We agree with the reviewer that localization uncertainty and integrated intensity can be a useful addition to the tracker output, and as a GitHub-based open-source project, we intend to extend the current version with a contribution from the community and our team. Regarding the current tool, the Radial Symmetry center approach in the implementation made available as a reference in the text only provides the standard deviation of the fitted 2D gaussian as an estimation, and not a calculated parameter, and does not include an integrated intensity. However, for the purposes of simple centroid determination, it provides an accurate and fast avenue. A cautionary statement regarding the use of the tool has been added to make the users aware of these characteristics of the tool. Simulations The displacement distribution was modified to use N(0,sqrt(2D dt)). numpy.random.randn returns a sample from the standard normal distribution N(0,1). To convert this to any normal distribution N(mu, sigma^2) => sigma * N(0,1) + mu. For mu=0 and var=sigma^2=2D dt, we indeed need to change the multiplicative factor in the free (Brownian) equation from sqrt(D dt) to sqrt(2 D dt). This modification was done in both the software and in the paper. Also, in the context of the movie simulator, we used the SNR parameter as a way to adjust the contrast between the simulated particle intensity and the background intensity (contrast = particle intensity – background intensity). To better represent this, we have renamed this parameter to “contrast” in the GUI, the code, the documentation, and the paper. Trait2d_analysis gui_gui: The warnings produced by the analysis GUI were the result of an unchecked bug. This has been fixed and no warnings should be produced now. We would like to thank the reviewer for bringing this to our attention. The “Max. fit iterations” parameter is internally passed along to the “maxfev” (maximum function evaluations) parameter of scipy.optimize.curve_fit, which is responsible for performing the actual model fits. In case fits fail, it might help to increase this parameter. If the “Use parameters as initial guesses” checkbox is checked, the output parameters of the last fit will be passed to the “p0” parameter of scipy.optimize.curve_fit and used as initial guesses for the next fit. Similar to “Max. fit iterations” this might be used to improve existing fits in some scenarios. The Range parameter is, indeed, equivalent to the fix_max_time argument present in the code at various points. The issue of proper range determination is already presented in the paper. A link to the paper was added to the README file in the repository, so that it can serve as further documentation for the user, together with the present reviewer report. Once again, we extend an invitation to the reviewer to write an issue on the GitHub repository on this topic, following the finalization of the review process, to direct further developments of the tool. Trait2d_simulator_gui: Thank you for these suggestions. We modified the default simulation parameters to make sure that the “Free diffusion” and “Hopping Diffusion” models can be used without necessitating too many resources. We added a dialog box to inform the user of the generated movie size before performing the movie simulation. Also, information about the simulation process is shown in the terminal. Trait2d_tracker_gui: Taking into account the reviewer suggestions, we now have updated the tracker with the following features: Import 16-bit TIFF stack as well as 8-bit Save/load parameters of the tracker Test run of the linking algorithm, where the user can select the frame range to test the tracking before processing the entire image sequence. The detection results can be viewed directly in the viewer (preview button). We have also provided an example stack with a parameter file, which can be found in the Github repository. Typos We thank the reviewer for pointing these out for our attention. We addressed and corrected all of them as advised. References: [1] M. Goulian and S. M. Simon, “Tracking Single Proteins within Cells,” Biophys. J., vol. 79, no. 4, pp. 2188–2198, Oct. 2000, doi: 10.1016/S0006-3495(00)76467-8. [2] G. Muñoz-Gil et al., “Objective comparison of methods to decode anomalous diffusion,” Nat. Commun., vol. 12, no. 1, 2021, doi: 10.1038/s41467-021-26320-w." } ] }, { "id": "95432", "date": "05 Oct 2021", "name": "Eva Christensen Arnspang", "expertise": [ "Reviewer Expertise Bioimaging", "membrane proteins", "nano domains", "cell organelles", "super-resolution microscopy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors describe a novel analysis code for analyzing SPT data allowing for short-term observations. The code allows for tracking even at fast acquisition rates, yields information about different diffusion modes, and includes a GUI. A very well-written paper on novel SPT analysis method. I should be very interested in running the code in my own lab.\nThe paper in its current form is acceptable for indexing, but it would enhance the reading experience for the reader with few well-chosen image examples of how the different components in the code improve the image analysis added, e.g. blurring, in addition to Figures 1 and 2. I would also recommend citing Mortensen et al., 20101 - in this paper, a similar analysis technique is described.\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": "7609", "date": "31 Jan 2022", "name": "Francesco Reina", "role": "Author Response", "response": "We thank the reviewer for this kind report. We look forward to the applications of our code in her research group and remain open to suggestions for further changes, optimizations, and support requests. We are not sure what the reviewer intends when suggesting how the code would improve image analysis. In fact, the code provided does not act on the time-lapse image stacks themselves in any part, save from a simple background subtraction pre-processing step in the tracker GUI, and does not add any blurring on top. The blurring correction added refers to the fact that the localization of a diffusing particle, when imaged in its motion, will be subject to additional errors, which can be corrected with a term dependent on the sampling rate of the measurement, the diffusion coefficient, and a coefficient R dependent on the illumination mode used by the optical setup [1]. In case the reviewer intended something else, we could provide some examples upon suggestion. Concerning the additional reference, we appreciate the suggested work, which is definitely a worthwhile read for every scientist seeking a comparison of methods for accurate single-particle localization. Therefore, we have added the suggested reference with appropriate text modifications." } ] } ]	1	https://f1000research.com/articles/10-838
https://f1000research.com/articles/10-380/v1	13 May 21	{ "type": "Research Article", "title": "Roles and Problems of Stroke Caregivers: A Qualitative Study in Yogyakarta, Indonesia", "authors": [ "Paryono Muhrodji", "Hendrawan Dian Agung Wicaksono", "Sekar Satiti", "Laksono Trisnantoro", "Ismail Setyopranoto", "Amelia Nur Vidyanti", "Paryono Muhrodji", "Hendrawan Dian Agung Wicaksono", "Sekar Satiti", "Laksono Trisnantoro", "Ismail Setyopranoto" ], "abstract": "Background: Caregivers play a central role in post-stroke patients care. However, the role of and problems managed by caregivers have not been widely studied, particularly in Indonesia. This study aims to explore the roles and problems of caregivers in post- stroke patients’ care. Method: This was a qualitative study. Caregivers of post-stroke patients from the homecare clinic of Dr Sardjito General Hospital were purposely selected during January 2017 to June 2018. Focus group discussions were conducted to explore the roles and problems of caregiving. Results: Themes related to caregivers’ roles were: connecting patients with medical personnel and other family members, maintaining patients’ health conditions by fulfilling basic needs and assisting rehabilitation, as well as maintaining patients’ psychological conditions by encouraging conversation, telling jokes, or recreation. On the other hand, themes related to caregivers’ problems were: lack of knowledge caused by education inadequacy, underappreciated and unconcerned family, suboptimal service including limited physiotherapy and pharmacy resource, unthorough administration, lack of communication, physical limitations, and burnout, as well as uncooperative patients. Conclusions: Caregivers play essential roles as communicators and help to maintain patient's health conditions. Common problems are related to a lack of knowledge about strokes and a lack of attention from family. Understanding the roles and problems of caregivers may help facilitate better management and increase the quality of life for both patients and their caregivers.", "keywords": [ "caregiver", "role", "problem", "homecare", "stroke", "post-stroke care" ], "content": "Introduction\n\nCaring for stroke survivors is a long-term experience that could influence a person’s quality of life.1 In Indonesia, stroke has been the leading cause of death and disability for ten consecutive years (2009-2019).2 Stroke, mainly affecting the elderly, may cause lifelong immobility, speech and communication problems, cognitive decline, urinary/fecal incontinence, and behavior changes that generally need long-term care.3,4 Therefore, stroke survivors become dependent on their caregivers, who take-on numerous roles and duties.3,5\n\nCaregivers play a central role in post-stroke patients’ care. Caregivers, which can be either family members or professional caregivers, are recommended to be involved in post-stroke patients care to improve the quality of patient management.6 Currently, there are vast studies related to the experiences of caring for post-stroke patients. However, the role and problems managed by the caregiver while caring for post-stroke patients in Indonesia have not been widely studied. Accordingly, this study aims to explore the roles and problems of post-stroke caregivers in Indonesia.\n\n\nMethods\n\nThis was a qualitative study to contextually explore a range of experiences and perspectives, not to seek an agreement. We used a grounded theory approach7 to identify initial themes and categories which describe the roles and problems of caregivers for post-stroke patients.\n\nWe recruited caregivers from the homecare clinic of Dr Sardjito General Hospital during January 2017 to June 2018, using purposive sampling. Inclusion criteria were: (1) family or professional caregivers, who caregiving a post-stroke patient with any level of disability; and (2) providing care for ≥1 year. We sent a checklist of the eligible criteria to the homecare clinic. Staff at the homecare clinic subsequently provided us with potential participants’ phone numbers with their consent. We contacted the potential participants through telephone calls to invite them to the study. A focus group discussion was scheduled after receiving confirmation of their participation. A total of seven caregivers were included in this study. Two caregivers refused to participate in the study due to personal reasons.\n\nData were collected by conducting a focus group discussion (FGD) on 21 July 2019. The research team developed an interview guide which had been further transformed into several questions to be addressed in the discussion. The discussion was led by a moderator (the second author HDAW), assisted by a note-taker and an assistant who documented the discussion. The first author (PM, a senior neurologist) was also present during the discussion to supervise and guarantee that the discussion went effortlessly. The moderator (interviewer) was a male senior neurology resident who had basic training skills of FGD. He did not have any relationships with the participants and the participants knew him as a neurology resident who had interest in research related to stroke caregivers.\n\nParticipants’ demographic data were collected at the beginning of the discussion, along with that of their care recipients’ (post-stroke patients). These data included age, sex, education level, main occupation, relationship with post-stroke patient, and years of caregiving. The discussion began with a broad central question regarding the caregivers’ knowledge about stroke followed by probing and further questions related to roles and problems of caregivers. The discussion was held in a quiet and comfortable closed room (workplace) for approximately 120 minutes. It was carried out once and there were no repeat sessions.\n\nAll discussions were tape-recorded and transcribed verbatim in Bahasa Indonesia. Data analysis began immediately after the discussion to identify ideas for generating categories. The semantic contents were separated and coded. Following a grounded theory approach, constant comparative analysis was used to compare data and open coding determined core categories. Identical codes were further grouped into categories to identify the main themes and subcategories.3 QDA Miner Lite version 2.0.6 software was used to analyse the transcript and assist the coding process. The coding was further reviewed by two investigators (PM and HDAW) to establish that codes were data-driven. Any differences were discussed until consensus was reached. The full transcript was not returned to participants for comment.\n\nThe study protocol received ethical approval from the Medical and Health Research Ethics Committee (Ethical approval reference number: KE/FK/0800/EC/2020) at the Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia.\n\nAll participants were given a brief explanation of the research and gave written informed consent in accordance with the Declaration of Helsinki.\n\n\nResults\n\nWe enrolled seven caregivers; all subjects were female with a median age of 36 years. All had home-based responsibilities and half of them had less than 5 years of caregiving experience. These caregivers managed five post-acute stroke patients. Patients’ median age was 72 years. Four (57.2%) of the caregivers had higher education while the other three (42.8%) had secondary education.\n\nFive caregivers (71.4%) were full-time carers, while two others (28.6%) still had another job. Most of the caregivers (57,2%) were professional caregivers, while the other three (42.8%) were family members. One of the caregivers (14.2%) had been working as a caregiver for more than 10 years, two caregivers (28.6%) had been working for 5-10 years, and four others (57.2%) less than five years. The basic characteristics of the participants are depicted in Table 1.\n\nAfter the discussion had been transcripted, 18 coding variables related to the roles of the caregiver for the post-stroke patient emerged from qualitative analysis. These 18 variables were reduced to six categories: communication, basic needs fulfillment, rehabilitation assistance, complication prevention and management, responsibility of patient’s condition, and maintenance of patient’s psychological condition. Furthermore, these six categories were reduced into three subthemes: communication, physical health, and patient’s psychology (Table 2).\n\nA total of 28 codes which related to physical activity were identified which were then reduced into seven categories: knowledge, family support, limited resources and facilities, lack of communication with medical personnel, communication problem with patient, refusal to treatment, and fatigue (burnout). These seven categories are further reduced into five major subthemes: knowledge, family support, sub-optimal service, communication, as well as physical and mental limitation (Table 2).\n\n\nDiscussion\n\nCaregivers act as a communicator between the patient or family and medical personnel, or the patients and their family. Caregivers connect communication between the patient and people involved in the patient’s management including doctors, psychologists, nurses, and therapists. This role serves as an important aspect of patient care, and also be integrated as a guideline for adult stroke rehabilitation and recovery.6,8 Therefore, good communication skills are necessary for caregivers.9\n\nAn example of this role can be seen in the following quote:\n\n“... I have all the nurses’ contact numbers and mmm... what is it called... doctors’. And usually, if there is any problem... last night, for instance, she was using an urinary catheter and also a diaper. However, when I checked it, the diaper had been full with urine. In the morning, I sent text messages to a nurse and reported it: 'This blah blah blah, please check the catheter as it may be leaking...” (Participant 6)\n\nCaregivers as the people who daily assist patients’ activities, hold responsibility for patients’ health care. Caregivers provide basic needs fulfillment and rehabilitation processes, as well as prevention and management of complications.6,9\n\nFor providing basic need fulfillment, caregivers help patients with disabilities in need of self-care and mobility such as feeding, bathing or showering, toileting, dressing, grooming, walking, and transferring. Caregivers also monitor the sleep pattern of their patients and help them to have good and adequate sleep.9,10\n\nDuring rehabilitation, caregivers also play a role in assiting rehabilitation processes by giving additional physiotherapy services at home. This is in accordance with the rehabilitation recommendations which state that families and caregivers are involved in determining the goals and implementation of post-stroke patient rehabilitation.8\n\nFurthermore, caregivers play essential roles in preventing and managing complications that may eventually develop. This role is in accordance with a previous study which found that caregivers can help reduce the incidence of post-stroke complications.11 However, the study above used a formal training program for caregivers which was too impractical to be implemented in this study. The importance of training and education for caregivers is also supported by other studies that showed a reduction in complications through providing education programs to caregivers.12\n\nThis role can be seen in the following quotes:\n\n“… yes, I stand by for 24 hours. At night I sometimes sleep next to him, to supervise him …” (participant 6)\n\n“… besides visiting physiotherapists in a hospital or inviting them to do home care services, in the morning and afternoon we usually have short additional physiotherapy session, only several minutes, just to stretch some muscles …” (participant 1)\n\n“… because my mother is ee .. her mobility is very limited, thus I need to feeding her using feeding tube. Other than that, what is it called? Catheter? Yes, a catheter! My mother is prone to urinary tract infection due to the catheter. Hence, sometimes if this infection develops, I have to... really have to do this, um ... to keep it clean... \"(participant 7)\n\nBased on the above discussion, it can be observed that maintaining the good health of post-stroke patients is the responsibility of the caregiver. This is in accordance with prior studies which stated that there are major changes in the responsibilities of families caring for post-acute stroke patients.13-15\n\nThis can be elaborated with the quote from one of the interviewees below:\n\n“... this is my responsibility that she is completely dependent on us ... so we take care of her like 'because she is already completely dependent on us, so whatever happens to her, in my mind, it counts on us'... ”(participant 6)\n\nCaregivers spend a considerable amount of time engaging with patients and contribute to maintaining patients’ psychological conditions.12 Based on the discussion, it was found that the caregivers help in maintaining patients’ psychological condition by telling interesting stories, joking, or doing recreational activities. This can be seen in the following statement:\n\n“… especially what we maintain is stabilitation of his mood. If he is happy, maybe mm... at least it doesn’t worsen his condition …” (participant 5)\n\nThe role of caregivers in maintaining patients’ psychological health conditions is important in preventing depression in post-acute stroke patients. Depression is one of the most common complications of stroke.16 Caregivers also have a great influence on the development of the patient's psychological condition and the prognosis.17\n\nAfter conducting the FGD, it was found that one of the problems in post-stroke patient care is caregivers’ lack of knowledge. Caregiver illiteracy on stroke was clearly seen during FGD. When the caregivers were asked ‘what is stroke’, one of them answered:\n\n“… I actually don’t know a thing about stroke, the definition and how …” (participant 6)\n\nThe other caregivers stated that they need to be equipped with some knowledge because for example, sometimes caregivers are involved in decision-making. However, they feel that they do not have sufficient knowledge to make a decision. This results in decision-making delay. The caregivers also feel that knowledge about medication and treatment is important. However, the information given to them by medical personnels is also limited. To overcome their lack of knowledge about stroke, they searched the information from other sources. However, the information obtained is said to be inaccurate, as in the statement below:\n\n“… sometimes it is confusing, you know. For example, in stroke management, some sources said it is okay to do acupuncture, some other said it’s not recommended, but... it's varying and confusing... \"(participant 2)\n\nLack of caregivers’ knowledge can be a weak point in post-stroke patients care. There have been many studies which stated that one of the important modalities that a caregiver must have is knowledge.6,18,19 In addition, it is recommended that the process of decision-making and treatment planning should involve both the family and the caregiver.6,20\n\nDuring the discussion, it was found that the family was not prepared for an emergency situation. This can delay early patient management. Meanwhile, the interviewee, as a caregiver, did not dare to take action before instruction had been given or a decision was made by the family, even though the patient was in an emergency condition. This occurred because the caregiver was afraid of being blamed, which can be seen in the following statement:\n\n“… we are afraid that later we will be blamed. I’ve experienced that. It’s like ‘that’s to bold’, or in Javanese ‘kok wani-wanine (how dare you?)’, even though we already did our best... \"(participant 3)\n\nAnother caregiver said that she had been scolded when she encountered similar situation, like in the phrase below:\n\n\"Even got scolded\" (participant 3)\n\nMoreover, the family did not give enough appreciation to the caregivers.\n\n\"no respect from the family\" (participant 2)\n\nLack of support by the family becomes a burden for the caregiver. Furthermore, the lack of family support in post-stroke patients care may reduce the quality of patient care. Family support has an important role in the patients’ quality of life.21\n\nThe main obstacle felt by the family is the lack of personnel or facilities offered by the home care unit. The interviewees stated that due to limited physiotherapy facilities and resources, the interviewees sought additional personnel from other resources. In addition, there were also some difficulties in getting the prescribed medication.\n\nWe also found that results of laboratory test delivered to family or caregiver were taking too much time. There was also a case of mistaken identity of a test result which was commented on by saying:\n\n\"I think it could be a fatal mistake\" (participant 7)\n\nThey also expressed problems in unthorough administration. These experiences can be seen in the following quotes:\n\n\"... I feel that the number of the physiotherapy sessions is not enough, but I know that they are really busy. We divide the resources, that's why we add from the third party...\" (participant 5)\n\n\"... sometimes I'm confused, for example, the doctor said, 'Ma'am, the lab result is good', 'which lab result, doc? I think it’s been a while since the last lab checks' …”(participant 6)\n\n“The administration, I think, still have many rooms for improvement … Some things have been claimed, have been written, but then written again …”(participant 7)\n\nLimited resources become a challenge itself because it raises the gap between the standard of care and real-world capacity of service, ranging from prevention to post-acute care.22\n\nPerceptions that were obtained from the caregivers were that there was a lack of communication between medical personnel causing uncollaborative patient management. They stated that this caused the patient's treatment to be time-consuming. Poor communication is associated with less effective services.6\n\nApart from communication problems with medical personnel, we also found that communication problems with patients became a burden for the caregiver. This is especially experienced with patients who have communication disorders. In long-term care setting, caregivers’ anxiety rates have decreased. Nonetheless, caregivers’ depression rates and burdens are still high.23\n\nThe problems above can be seen in the following quote:\n\n“… for example, the physiotherapist came and asked about the patient’s development, then I explained ‘Oh she is blah, blah, blah’, ‘Okay, I will report it to the doctor’. Later, the doctor will come empty-handed … seems like uninformed. That is what sometimes makes us disappointed …”(participant 7)\n\nBased on the FGD, one of the obstacles in post-stroke patients care was patients’ refusal of treatment. The caregivers thought the patient's morale, pain, and burnout were the causes of the refusal. Apart from the patient side, caregivers also face physical and mental burnout.\n\nDepression is one of the complications that often occurs in patients with a history of stroke.16 Periodic examinations or screening related to depression in the patients and their caregivers by their doctor is recommended.6 In addition, the deteriorating psychological condition of the patient is a heavy burden for the caregiver.18 Anxiety and depression while caring the stroke patients are related to the caregivers’ burden.24\n\nLong working hours, quality of patient health, stroke severity, and level of patient dependency are factors associated with caregiver’s burnout.25 Other studies have shown that a high need for care is also a factor that influences caregiver’s burnout.26 Moreover, the emotional condition of the patient also affects caregiver’s burnout.27 The description of this problem can be seen in the following quote:\n\n\"... during speech therapy, she persistently refused the therapy. The therapy ... at that time, it was from ... the speech therapy from Sardjito Hospital. When the therapist came, she pretended to sleep, and later after the therapist had came home, she woke up. She constantly refused it, she kept looking away. 'Ma'am, the therapist is coming ...' she insisted she did not want it. For communication, she responded, but she refused the therapy... \"(participant 7)\n\nThis study has several limitations. First, the caregivers in this study were recruited from one medical center, and the sample size was small. Second, our findings might not be generalized to non-Asian countries as our participants were recruited in Indonesia which has collectivist backgrounds.28 Third, all the caregivers in our study were female which may cause differences in caregiving experiences and lead to different roles and problems. Future study can be conducted with male caregivers to capture holistic understanding of caregivers’ role and problems.\n\n\nConclusions\n\nCaregivers play an essential role in connecting patients with medical personnel and other family members, as well as maintaining the patient's physical and psychological condition. Meanwhile, caregivers face the problems of lack of support from patients' family, lack of knowledge, suboptimal service, lack of communication and caregivers’ burnout. The recognition of these roles and problems may help facilitate better management of post-stroke patients and increase quality of life for both patients and their caregivers.\n\n\nData availability statement\n\nZenodo: Roles and problems of stroke caregivers: A qualitative study in Yogyakarta, Indonesia. https://doi.org/10.5281/zenodo.4716264.29\n\nThe project contains the following underlying data:\n\n• English Translated Transcript-Edited-Final.doc (an English translation of the focus group transcript).\n\n\nReporting guidelines\n\nZenodo. Roles and problems of stroke caregivers: A qualitative study in Yogyakarta, Indonesia. https://doi.org/10.5281/zenodo.4729367.\n\nThe project contains the following reporting checklist:\n\n• COREQ_checklist.pdf\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\nMuhrodji P: writing-original draft preparation, conceptualization, methodology, formal analysis; Wicaksana HAD: data curation, investigation; Satiti S: supervision, validation; Trisnantoro L: conceptualization, supervision, validation; Setyopranoto I: conceptualization, supervision, validation; Vidyanti AN: visualization, validation, writing-review & editing.", "appendix": "Acknowledgements\n\nThe authors would like to extend their gratitude to all the patients and caregivers who participated in this study. We would also thank all the staffs at Home Care Service in Dr. Sardjito General Hospital for their technical assistance.\n\n\nReferences\n\nLynch EB, Butt Z, Heinemann A, et al.: A qualitative study of quality of life after stroke: the importance of social relationships. J Rehab Med. 2008; 40: 518–523.\n\nInstitute for Health Metrics and Evaluation: Global Burden of Disease: Indonesia. 2020.\n\nTseng C-N, Huang G-S, Yu P-J, et al.: A qualitative study of family caregiver experiences of managing incontinence in stroke survivors. PLoS One. 2015; 10: e0129540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVirani SS, Alonso A, Benjamin EJ, et al.: Heart disease and stroke statistics—2020 update: a report from the American Heart Association. Circulation. 2020; 141: e139–e596. PubMed Abstract \| Publisher Full Text\n\nKing RB, Ainsworth CR, Ronen M, et al.: Stroke caregivers: pressing problems reported during the first months of caregiving. J Neurosci Nurs. 2010; 42: 302–311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuncan PW, Zorowitz R, Bates B, et al.: Management of adult stroke rehabilitation care: a clinical practice guideline. Stroke. 2005; 36: e100–e143. PubMed Abstract \| Publisher Full Text\n\nCorbin J, Strauss A: Basics of qualitative research. 3rd ed Thousand Oaks, CA: Sage; 2008.\n\nWinstein CJ, Stein J, Arena R, et al.: Guidelines for adult stroke rehabilitation and recovery: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2016; 47: e98–e169. PubMed Abstract \| Publisher Full Text\n\nLau DT, Kasper JD, Hauser JM, et al.: Family caregiver skills in medication management for hospice patients: a qualitative study to define a construct. J Gerontol B Psychol Sci Soc Sci. 2009; 64: 799–807. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Academies of Sciences Engineering and Medicine: Families caring for an aging America. Washington DC: National Academies Press; 2016.\n\nPitthayapong S, Thiangtam W, Powwattana A, et al.: A community based program for family caregivers for post stroke survivors in Thailand. Asian Nurs Res (Korean Soc Nurs Sci). 2017; 11: 150–157. PubMed Abstract \| Publisher Full Text\n\nZhang L, Zhang T, Sun Y: A newly designed intensive caregiver education program reduces cognitive impairment, anxiety, and depression in patients with acute ischemic stroke. Braz J Med Bio Res. 2019; 52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLutz BJ, Ellen Young M, Cox KJ, et al.: The crisis of stroke: experiences of patients and their family caregivers. Top Stroke Rehabil. 2011; 18: 786–797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLutz BJ, Young ME, Creasy KR, et al.: Improving stroke caregiver readiness for transition from inpatient rehabilitation to home. Gerontologist. 2017; 57: 880–889. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoon M: The unprepared caregiver. Gerontologist. 2017; 57: 26–31. PubMed Abstract \| Publisher Full Text\n\nCaplan LR: Caplan's stroke. Cambridge University Press; 2016.\n\nZhao F-Y, Yue Y-Y, Li L, et al.: Clinical practice guidelines for post-stroke depression in China. Braz J Psychiatry. 2018; 40: 325–334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurcotte M: Family caregiving: What are the consequences? In: Statistics Canada. (ed.). Canada: Minister of Industry; 2013.\n\nDharma KK, Damhudi D, Yardes N, et al.: Increase in the functional capacity and quality of life among stroke patients by family caregiver empowerment program based on adaptation model. Int J Nurs Sci. 2018; 5: 357–364. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu C, Marino V, Sheehan OC, et al.: Association between caregiver engagement and patient-reported healthcare utilization after stroke: a mixed-methods study. Top Stroke Rehabil. 2020; 27: 1–7. PubMed Abstract \| Publisher Full Text\n\nRahman R, Dewi FST, Setyopranoto I: Dukungan keluarga dan kualitas hidup penderita stroke pada fase pasca akut di Wonogiri. Berita Kedokteran Masyarakat. 2017; 33: 383–390. Publisher Full Text\n\nJohnson W, Onuma O, Owolabi M, et al.: Stroke: a global response is needed. Bull World Health Organ. 2016; 94: 634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKruithof WJ, Post MW, van Mierlo ML, et al.: Caregiver burden and emotional problems in partners of stroke patients at two months and one year post-stroke: Determinants and prediction. Patient Educ Couns. 2016; 99: 1632–1640. PubMed Abstract \| Publisher Full Text\n\nHu P, Yang Q, Kong L, et al.: Relationship between the anxiety/depression and care burden of the major caregiver of stroke patients. Medicine. 2018; 97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOliva-Moreno J, Peña-Longobardo LM, Mar J, et al.: Determinants of informal care, burden, and risk of burnout in caregivers of stroke survivors: the CONOCES study. Stroke. 2018; 49: 140–146. PubMed Abstract \| Publisher Full Text\n\nYang JO, Lee HK: Factors influencing burnout in primary family caregivers of hospital-based home care patients. J Korean Acad Community Health Nurs. 2018; 29: 54–64. Publisher Full Text\n\nFerro JM, Santos AC: Emotions after stroke: A narrative update. Int J Stroke. 2020; 15: 256–267. PubMed Abstract \| Publisher Full Text\n\nIrawanto D: An Analysis Of National Culture And Leadership Practices In Indonesia. J Divers Manag. 2009; 4: 41–48. Publisher Full Text\n\nParyono W, Hendrawan DA, Satiti S, et al.: Roles and problems of stroke caregivers: A qualitative study in Yogyakarta, Indonesia (Version dataset) [Data set]. Zenodo. 2021. Publisher Full Text" }	[ { "id": "94919", "date": "05 Oct 2021", "name": "Aznida Firzah Abdul Aziz", "expertise": [ "Reviewer Expertise Family Medicine", "Health system Research", "Community Stroke & Economic evaluations" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: Roles and Problems of Stroke caregivers: A Qualitative Study in Yogyakarta, Indonesia.\nAbstract: Satisfactory.\nGeneral impression: This is a novel attempt to document the roles and problems of stroke caregivers in Indonesia. The methodology is sound, but the manuscript would benefit from a more detailed description and discussion of the subject matter. The study setting needs to be introduced in greater detail as caregivers’ roles are very much affected by healthcare system differences across the world. Post stroke care is very challenging particularly for the LMIC, and especially for countries in the ASEAN region. Caregiver definitions in the Asian perspective also differ from western literature - and this should be highlighted in the write up. The authors may refer to this article (which I was involved with) by Ozdalifah O et al. (2021) 1 for definitions of stroke caregivers.\nOverall, this manuscript has potential to be accepted after major revision/refinement.\nIntroduction: This section was too brief (2 paragraphs) and did not highlight the caregiver situation in Indonesia (i.e. what is the norm in post stroke care, and who constitutes as caregiver?). There is mention that caregivers are made up of family members and professional caregivers. Suggest to highlight and define what professional caregivers are (i.e. salaried professional nurse/ therapist/trained domestic helper etc.) and where were the caregivers located - is it in the patients’ own home or at a inpatient rehabilitation facility/ nursing home?\nMethod: Focus group discussion was conducted to explore the roles and problems of caregiving (only ONE FGD was conducted, as mentioned in the article).\nSample recruitment: \"Inclusion criteria were: (1) family or professional caregivers, who caregiving a post-stroke patient with any level of disability; and (2) providing care for ≥1 year.\" Comment: The definition of professional caregivers is unclear. Do you mean professional caregivers as trained and/or were they salaried caregivers?\nData analysis: Why was the transcription not distributed to the caregivers for checking, verification? Is there a specific reason for this?\nResults: All subjects (caregivers) were female, median age of 36 years (?IQR), patients’ median age was 72 years (?IQR).\nDiscussion: Roles of caregiver: Communication: The areas where communication problems most likely to occur should be grouped, summarized and examples quoted accordingly. Suggest to include examples of communication between:\nCaregiver and patient; Caregiver and medical personnel i.e. doctor; Therapists and doctor.\nRole of caregiver: maintain patients’ health: Similarly, the flow of this section can be improved by citing the quotes after each point. Please maintain a consistent format throughout the results section. Below are suggested rewrites:\nParagraph 2:\n“For providing basic need fulfilment, caregivers help patients with disabilities in need of self-care and mobility such as feed, bathing, showering, toileting, dressing, grooming, walking and transferring…”\n\n“Caregivers also monitor the sleep pattern of their patients and help them to have good and adequate sleep.”\n\n“….yes, I standby for 24 hours. At night I sometimes sleep next to him, to supervise him…’(participant 6, ?family/professional caregiver)\nParagraph 3.\n“During rehabilitation, caregivers play a role in assisting rehabilitation processes by giving additional physiotherapy services at home. This is in accordance with the rehabilitation recommendations that families and caregivers are involved in determining the goals and implementation of post-stroke rehabilitation.8”\n\n“..besides visiting physiotherapists in a hospital or inviting them to do home care services, in the morning and afternoon we usually have short additional physiotherapy session, only several minutes, just to stretch some muscles….” (participant 17, family / professional caregiver).\n\nCaregivers’ problems: unappreciated and unconcerned family.\n\n“Moreover, the family did not give enough appreciation to the caregivers.\n\n“no respect from the family” (participant 2, ?professional/family caregiver)\nConclusion: This section should summarise and provide recommendations on how to handle the caregiver issues. E.g. identifying relevant information which should be passed on to the identified caregiver: information regarding stroke; overall management/rehabilitation plans; communication strategies between caregiver-therapist-doctor; therapist-caregiver; professional caregiver-family; caregiver respite issues (teaching caregivers on how to handle or cope with burnout, for professional caregivers to be given scheduled respite hours/days).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7746", "date": "31 Jan 2022", "name": "Amelia Nur Vidyanti", "role": "Author Response", "response": "Title: Roles and Problems of Stroke Caregivers: A Qualitative Study in Yogyakarta, Indonesia. Response to reviewer 1. We wish to thank you all for your constructive comments in this review. Your comments provided valuable insights to refine its contents. Because one of the objectives of this study is to widen our knowledge about caregivers’ roles and problems, especially in Indonesia, we would like to refine our paper. As you advise us to, more detail about this study setting and definitions about caregivers which enrolled in this study have been added. Comment 1 Introduction: This section was too brief (2 paragraphs) and did not highlight the caregiver situation in Indonesia (i.e. what is the norm in post stroke care, and who constitutes as caregiver?). There is mention that caregivers are made up of family members and professional caregivers. Suggest to highlight and define what professional caregivers are (i.e. salaried professional nurse/ therapist/trained domestic helper etc.) and where were the caregivers located - is it in the patients’ own home or at a inpatient rehabilitation facility/ nursing home? Response: We would like to thank you all for this comment. We agree that the differences in who cares for stroke patients (nurse or nonmedical-trained person) should be included in introduction to give our readers a bit of knowledge about how stroke patients are being cared in Indonesia. We have also added a reference as you suggest in refer to the definition of caregivers in Asian population. Revised text : Caregivers can be either formal or informal caregivers6. Formal care is delivered by professionals, such as nurses, physiotherapists, or speech and occupational therapists. Informal caregivers can be paid or unpaid. Unpaid informal caregivers are family members, friends, or relatives, meanwhile paid informal ones are domestic helpers or trained individuals6. Caregiving services can be provided in a home-based setting, a rehabilitation facility, or a nursing home7.In Asian population, the responsibility of caring the patients mostly relies on family members, particularly the spouse or offspring who can provide caregiving in home-bound setting8. [ANV1] Comment 2 Method: Focus group discussion was conducted to explore the roles and problems of caregiving (only ONE FGD was conducted, as mentioned in the article). Sample recruitment: \"Inclusion criteria were: (1) family or professional caregivers, who caregiving a post-stroke patient with any level of disability; and (2) providing care for ≥1 year.\" Comment: The definition of professional caregivers is unclear. Do you mean professional caregivers as trained and/or were they salaried caregivers? Response: We thank for your valuable comment. In this study, we recruited only informal caregivers. We apologize by making a mistake in mentioning professional caregivers. Hence, we have revised the text as below: Inclusion criteria were: (1) family caregivers or a domestic helper[hd2] ,. Comment 3 Data analysis: Why was the transcription not distributed to the caregivers for checking, verification? Is there a specific reason for this? Response: There was no specific reason for not returning the transcript to respondents. We realize that it may lead to bias or misinterpretation, thus we put this issue in the limitation. We will take this point as a suggestion for improvement in further research. Comment 4 Results: All subjects (caregivers) were female, median age of 36 years (?IQR), patients’ median age was 72 years (?IQR). Response: Initially we only used median, minimum, and maximum number due to the sample size for this study is small. However, we have added the interquartile range as well in the revised version as below: We enrolled seven caregivers; all subjects were female with a median age of 36 years (interquartile range [IQR] was 17)[ANV3] . All had home-based responsibilities and half of them had less than 5 years of caregiving experience. These caregivers managed five post-acute stroke patients. Patients’ median age was 72 years (IQR was 14)[ANV4] . Four (57.2%) of the caregivers had higher education while the other three (42.8%) had secondary education. Comment 5 Discussion: Roles of caregiver: Communication: The areas where communication problems most likely to occur should be grouped, summarized and examples quoted accordingly. Suggest to include examples of communication between: Caregiver and patient; Caregiver and medical personnel i.e. doctor; Therapists and doctor. Response: Communication problems had been found in caregivers’ problems’ section. We have rewritten this section as you suggest us to. Communication problems between therapists and doctors, while were very likely to occur, were not surfaced during FGD. There were some other themes that likely been caused by this particular problem (therapists and doctor’s communication), however we’re afraid that were just our assumption so we did not include this in our writing. Despite of this, we have re-organized the communication problems and added one more quote to describe related to communication problem between caregivers and the patients: Caregivers’ problems: lack of communication Perceptions that were obtained from the caregivers were that there was a lack of communication between medical personnel causing uncollaborative patient management. They stated that this caused the patient's treatment was to be time-consuming. Poor communication is associated with less effective services7. The problems above can be seen in the following quote: “…for example, the physiotherapist came and asked about the patient’s development, then I explained ‘Oh she is blah, blah, blah’, ‘Okay, I will report it to the doctor’. Later, the doctor will come empty-handed…seems like uninformed. That is what sometimes makes us disappointed… ”(participant 7) [hd5] Apart from communication problems with medical personnel, we also found that communication problems with patients became a burden for the caregiver. This is especially experienced with patients who have communication disorders. In long-term care setting, caregivers’ anxiety rates have decreased. Nonetheless, caregivers’ depression rates and burdens are still high25. This was an example which expressing this problem: “… after her second stroke she really was deteriorated, we didn’t know whether she still recognized us or not because there were no response at all from her, she was only staring but it’s a blank stare…”[hd6] Comment 6 Role of caregiver: maintain patients’ health: Similarly, the flow of this section can be improved by citing the quotes after each point. Please maintain a consistent format throughout the results section. Below are suggested rewrites: Paragraph 2: “For providing basic need fulfilment, caregivers help patients with disabilities in need of self-care and mobility such as feed, bathing, showering, toileting, dressing, grooming, walking and transferring…” “Caregivers also monitor the sleep pattern of their patients and help them to have good and adequate sleep.” “….yes, I standby for 24 hours. At night I sometimes sleep next to him, to supervise him…’(participant 6, ?family/professional caregiver) Paragraph 3. “During rehabilitation, caregivers play a role in assisting rehabilitation processes by giving additional physiotherapy services at home. This is in accordance with the rehabilitation recommendations that families and caregivers are involved in determining the goals and implementation of post-stroke rehabilitation.8” “..besides visiting physiotherapists in a hospital or inviting them to do home care services, in the morning and afternoon we usually have short additional physiotherapy session, only several minutes, just to stretch some muscles….” (participant 17, family / professional caregiver). Response: We have re-organized this section by citing the quote after each point in order to improve the flow of writing. Roles of caregiver: maintaining patients’ health Caregivers as the person people who daily assist patients’ activities, hold responsibility for patients’ health care. Caregivers provide basic needs fulfillment, and rehabilitation processes, as well as prevention and management of complications7,11 . For providing basic need fulfillment, caregivers help patients with disabilities in need of self-care and mobility such as feeding, bathing or showering, toileting, dressing, grooming, walking, and transferring. This quote below is an example from participant about this role: “…within a day, our services are bathing, when it is eating time we feed them, and.. usually Mr M is simple, after bathing we feed him via PEG…” (participant 4)[hd7] Caregivers[ANV8] also monitor the sleep pattern of their patients and help them to have good and adequate sleep11,12 . We can see this role in this quote: “…yes, I stand by for 24 hours. At night I sometimes sleep next to him, to supervise him…” (participant 6)[hd9] During rehabilitation, caregivers also play a role in assisting rehabilitation processes by giving additional physiotherapy services at home. This is in accordance with the rehabilitation recommendations which stated that families and caregivers are involved in determining the goals and implementation of post-stroke patient rehabilitation10. Role of caregiver in helping rehabilitation can be seen in this quote: “…besides visiting physiotherapists in a hospital or inviting them to do home care services, in the morning and afternoon we usually have short additional physiotherapy session, only several minutes, just to stretch some muscles…” (participant 1)[hd10] Furthermore, caregivers plays essential roles to in preventing and managing complications that eventually may eventually develop. This role is in accordance with a previous study which found that caregivers can help reduce the incidence of post-stroke complications13. However, the study above used a formal training program for caregivers which was rather too impractical to be implemented in this study. The importance of training or and education for caregivers is also supported by other studies that showed a reduction in complications through providing education programs to caregivers14 . This[ANV11] role can be seen in the following quote: “…because my mother is ee .. her mobility is very limited, thus I need to feeding her using feeding tube. Other than that, what is it called? Catheter? Yes, a catheter! My mother is prone to urinary tract infection due to the catheter. Hence, sometimes if this infection develops, I have to... really have to do this, um ... to keep it clean... \"(participant 7) Based on the above discussion, it can be observed that maintaining a the good health of post-stroke patients is the responsibility of the caregiver. This is in accordance with prior studies which stated that there are major changes in the responsibilities of families caring for post-acute stroke patients15-17 . This can be elaborated with the quote from one of the interviewees below: \"... this is my responsibility that she is completely dependent on us ... so we take care of her like 'because she is already completely dependent on us, so whatever happens to her, in my mind, it counts on us'... \"(participant 6) Comment 7 Caregivers’ problems: unappreciated and unconcerned family. “Moreover, the family did not give enough appreciation to the caregivers. “no respect from the family” (participant 2, ?professional/family caregiver) Response: We appreciate reviewer’s comment related this matter. Caregivers’ problems related to unappreciated and unconcerned family were the problems faced by domestic helpers as the informal caregivers. Therefore, we have written additional explanation in the quotes of participant 2 and 3, as written in the revised text. During the discussion, it was found that the family was not prepared for an emergency situation. This can delay early patient management. Meanwhile, some interviewees, domestic helpers being as informal caregivers[ANV12] , did not dare to take action before instruction had been given or a decision was made by the family, even though the patient was in an emergency condition. This occurred because the caregiver was afraid of being blamed, which can be seen in the following statement: “…we are afraid that later we will be blamed. I’ve experienced that. It’s like ‘that’s to bold’, or in Javanese ‘kok wani-wanine (how dare you?)’, even though we already did our best... \"(participant 3, a domestic helper[ANV13]) Another caregiver said that she had been scolded when she encountered similar situation, like in the phrase below: \"Even got scolded\" (participant 3, a domestic helper[ANV14]) Moreover, the family did not give enough appreciation to the caregivers. \"no respect from the family\" (participant 2, a domestic helper[ANV15]) Comment 8 Conclusion: This section should summarize and provide recommendations on how to handle the caregiver issues. E.g. identifying relevant information which should be passed on to the identified caregiver: information regarding stroke; overall management/rehabilitation plans; communication strategies between caregiver-therapist-doctor; therapist-caregiver; professional caregiver-family; caregiver respite issues (teaching caregivers on how to handle or cope with burnout, for professional caregivers to be given scheduled respite hours/days). Response: We appreciate your suggestions. However, we’re afraid that providing recommendation to handle these issues is out of scope of this paper. Maybe we can add some opinion to add some solution to these issues, but as the aim of this paper is to widen and deepen our knowledge about roles and problems of caregivers’, we decided not to add recommendation on how to handle the caregiver issues. Nevertheless, we have added a closing statement in the conclusion as below: Further research to study the effects of these findings on the quality of life of both patient and their caregiver, as well as how to handle the caregiver issues should be investigated.[hd16] [ANV1] As reviewer 1 suggested, we add more explanation about caregivers and the setting/location of care provided [hd2] As reviewer 1 asked, we clarify the type of caregivers recruited [ANV3] As reviewer 1 suggested, we add the IQR [ANV4] As reviewer 1 suggested, we add the IQR [hd5] The quote has been organized and moved right after the first paragraph [hd6] As reviewer 1 suggested, we add one more quote related to communication problem between caregivers and the patients [hd7] We add one more quote to provide more example related to the role of caregivers for providing basic needs [ANV8] As reviewer 1 suggested, we have moved citing the quote after each point to improve the flow of writing [hd9] As reviewer 1 suggested, we have moved citing the quote after each point to improve the flow of writing [hd10] As reviewer 1 suggested, we have moved citing the quote after each point to improve the flow of writing [ANV11] As reviewer 1 suggested, we have moved citing the quote after each point to improve the flow of writing [ANV12] Additional explanation related to the status of caregivers as domestic helpers [ANV13] Additional explanation related to the status of caregivers as domestic helpers [ANV14] Additional explanation related to the status of caregivers as domestic helpers [ANV15] Additional explanation related to the status of caregivers as domestic helpers [hd16] Usulan revisi kesimpulan sesuai dengan saran reviewer 2" } ] }, { "id": "94920", "date": "29 Oct 2021", "name": "Thuan Duc Nguyen", "expertise": [ "Reviewer Expertise Stroke", "Clinical Neurology especially Pain", "Movement disorders." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStroke is the leading cause of disability worldwide and has become a socioeconomic burden for our society. Caring for stroke survivors can play a crucial role in their continuous recovery in which caregiver is indispensable to these activities. The study mentioned roles and problems of post-stroke caregivers, which is meaningful in clinical practice. The manuscript is well-written, presents the research contents in detail as well as analyzes, synthesizes and draws out results consistent with the research objectives. However, there are a few points the authors also need to pay attention to:\nData collection needs to be very accurate and avoid interference because the data here are completely subjective information of the research object. For example, participant 6 said \"…yes, I stand by for 24 hours. At night I sometimes sleep next to him, to supervise him…\" - Is it really accurate to be with the patient 24 hours a day? When will the caregiver sleep?\n\nThe study sample size is too small to draw general representative results. A very important problem related to caregiver is the lack of sympathy, sharing and love for the patients. They take care of patients after stroke often for the sake of salary and purely for responsibility. Is this problem encountered in this study?\n\nOf the caregiver's roles and problems, which one is the most important? Can the authors rank the importance of those roles and issues?\n\nThe research objective only mentions the roles and problems of caregivers, so the conclusion should only focus on this point, not giving the opinion \"Understanding the roles and problems of caregivers may help facilitate better management and increase the quality of life for both patients and their caregiver”. The author can suggest and suggest some points for further research in the future, which is more reasonable.\nI hope the authors can edit, supplement and add their own discussion to make the study even more complete. This is really an interesting study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7747", "date": "31 Jan 2022", "name": "Amelia Nur Vidyanti", "role": "Author Response", "response": "Title: Roles and Problems of Stroke Caregivers: A Qualitative Study in Yogyakarta, Indonesia. Response to reviewer 2 We wish to thank you all for your constructive comments in this review. Your comments provided valuable insights to refine its contents. Comments Stroke is the leading cause of disability worldwide and has become a socioeconomic burden for our society. Caring for stroke survivors can play a crucial role in their continuous recovery in which caregiver is indispensable to these activities. The study mentioned roles and problems of post-stroke caregivers, which is meaningful in clinical practice. The manuscript is well-written, presents the research contents in detail as well as analyzes, synthesizes and draws out results consistent with the research objectives. However, there are a few points the authors also need to pay attention to: Data collection needs to be very accurate and avoid interference because the data here are completely subjective information of the research object. For example, participant 6 said \"…yes, I stand by for 24 hours. At night I sometimes sleep next to him, to supervise him…\" - Is it really accurate to be with the patient 24 hours a day? When will the caregiver sleep? Response: Thank you for your question. To clarify this quote, this statement from the participant was to depict that her duty was to standby for 24 hours. Of course, she did not wake up all day for 24 hours for monitoring and helping her patient. However, she meant that she was standby to caring her patient. The study sample size is too small to draw general representative results. A very important problem related to caregiver is the lack of sympathy, sharing and love for the patients. They take care of patients after stroke often for the sake of salary and purely for responsibility. Is this problem encountered in this study? Response: The caregivers’ problem we found in the present study is not lack of sympathy, sharing and love for the patient, but more to lack of respect between caregivers and (other) family members. The different findings may be due to different definitions of caregivers in Asian population. We mentioned this in the revised version in line 64-72. In Asian population, the responsibility of caring the post-stroke patients mostly relies on family members, particularly the spouse or offspring who can provide caregiving in home-bound setting. Of the caregiver's roles and problems, which one is the most important? Can the authors rank the importance of those roles and issues? Response: Thank you for your question, but we’re afraid that we cannot rank these roles and problems as we did not collect any quantitative data about these issues. We aim to identify the roles and problems of caregiving qualitatively. The research objective only mentions the roles and problems of caregivers, so the conclusion should only focus on this point, not giving the opinion \"Understanding the roles and problems of caregivers may help facilitate better management and increase the quality of life for both patients and their caregiver”. The author can suggest and suggest some points for further research in the future, which is more reasonable. Response: We would like to refine our conclusions. We agree that by understanding roles and problems of caregiver does not automatically improve post-acute care of stroke patient. Here is the revised conclusion: Conclusions Caregivers play an essential role in connecting patients with medical personnel and other family members, as well as maintaining the patient's physical and psychological condition. Meanwhile, the problems faced by caregivers face the problems of were lack of support from patients' family, lack of knowledge, suboptimal service, lack of communication and caregivers’ burnout. Further research to study the effects of these findings on the quality of life of both patient and their caregiver, as well as how to handle the caregiver issues should be investigated.[hd1] [hd1] [hd1] Revised conclusion as reviewer 2 suggested." } ] } ]	1	https://f1000research.com/articles/10-380
https://f1000research.com/articles/11-124/v1	31 Jan 22	{ "type": "Software Tool Article", "title": "SPARClink: an interactive tool to visualize the impact of the SPARC program", "authors": [ "Sanjay Soundarajan", "Sachira Kuruppu", "Ashutosh Singh", "Jongchan Kim", "Monalisa Achalla", "Sachira Kuruppu", "Ashutosh Singh", "Jongchan Kim", "Monalisa Achalla" ], "abstract": "The National Institutes of Health (NIH) Stimulating Peripheral Activity to Relieve Conditions (SPARC) program seeks to accelerate the development of therapeutic devices that modulate electrical activity in nerves to improve organ function. SPARC-funded researchers are generating rich datasets from neuromodulation research that are curated and shared according to FAIR (Findable, Accessible, Interoperable, and Reusable) guidelines and are accessible to the public on the SPARC data portal. Keeping track of the utilization of these datasets within the larger research community is a feature that will benefit data-generating researchers in showcasing the impact of their SPARC outcomes. This will also allow the SPARC program to display the impact of the FAIR data curation and sharing practices that have been implemented. This manuscript provides the methods and outcomes of SPARClink, our web tool for visualizing the impact of SPARC, which won the Second prize at the 2021 SPARC FAIR Codeathon. With SPARClink, we built a system that automatically and continuously finds new published SPARC scientific outputs (datasets, publications, protocols) and the external resources referring to them. SPARC datasets and protocols are queried using publicly accessible REST application programming interfaces (APIs, provided by Pennsieve and Protocols.io) and stored in a publicly accessible database. Citation information for these resources is retrieved using the NIH reporter API and National Center for Biotechnology Information (NCBI) Entrez system. A novel knowledge graph-based structure was created to visualize the results of these queries and showcase the impact that the FAIR data principles can have on the research landscape when they are adopted by a consortium.", "keywords": [ "Visualization", "machine-learning", "citations", "FAIR", "data sharing" ], "content": "Introduction\n\nThe National Institutes of Health (NIH) Common Fund’s Stimulating Peripheral Activity to Relieve Conditions (SPARC) program aims to transform our understanding of nerve-organ interactions with the intent of advancing bioelectronic medicine towards treatments that change lives.1 The SPARC program employs a Findable, Accessible, Interoperable, and Reusable (FAIR) first approach for its datasets, protocols, and publications, hence enabling the data to be easily reused by research communities globally. The SPARC data portal can be used as the gateway to access fully curated datasets at any time.2 Using the portal, researchers can search for data used in real-world experiments to verify or corroborate studies in device development. There is also potential for the data generated by the SPARC program to be useful outside the current field of study showcasing the benefits of multi-discipline data generation and sharing.3\n\nAll SPARC datasets are curated by the researchers according to the SPARC Data Standards (SDS), a data and metadata structure derived from the Brain Imaging Data Structure (BIDS).4 Several resources are made available to SPARC researchers for making their data FAIR, such as the cloud data platform Pennsieve, the curated vocabulary selector and annotation platform SciCrunch, the open-source computational modeling platform o2S2PARC, the online microscopy image viewer Biolucida, and the data curation software SODA.4–6 The datasets submitted by researchers also follow an extensive curation process where teams from the SPARC Data Resource Center (DRC) examine the submitted data and work with the researchers to ensure all aspects of the FAIR data principles are being followed.4,6,7 Once these datasets are made public, access to them is provided through the Pennsieve Discover service and sparc.science, the official access point of the SPARC Portal.8\n\nWhile the submission and curation of data are simplified by such tools, one of the greater benefits of the FAIR guidelines is the ability to reuse data in other studies by other researchers around the world. However, a researcher who has submitted a dataset might not always be aware of the reuse of their original submitted data since current citation indexing tools, like Google Scholar, do not account for datasets. To address this shortcoming, we developed SPARClink during the 2021 SPARC FAIR Codeathon (July 12th, 2021 – July 26th, 2021),9 a system that queries all external publications using open source tools and platforms and creates a database and visualizations of citations that are helpful to showcase the impact of the SPARC consortium. In this instance, we define impact as the frequency of citations of SPARC-funded resources. By using citations as the key measure in SPARClink, we have created a method for showcasing the reuse of generated data and the benefits that FAIR data generation practices have on the overall scientific community. A visual representation of the reuse of data will allow both researchers and the general public to see the benefits of the concept of FAIR data and the immediate utilization of publicly funded datasets in advancing the field of bioelectronic medicine.\n\n\nMethods\n\nOur solution can broadly be categorized into four steps. The first step involves the backend extraction of data using various application programming interfaces (APIs). The second step is setting up and storing the extracted data on a real-time database. The third step involves using machine learning to improve user experience by developing context-sensitive word clouds and smart keyword searches in the portal. The final step is used to create an engaging visualization that users of the SPARClink system will be able to interact with to view the extracted data. A visual representation of this workflow is shown in Figure 1.\n\nWe used the dataset information retrieved directly from the Pennsieve data storage platform by running the Pennsieve API to gather all publicly available SPARC datasets.10 The protocols stored on Protocols.io under the SPARC group were also queried via this method.11 A list of public and published DOIs was created in our database with additional information regarding the study authors and descriptions.\n\nWe used NIH RePORTER to retrieve data about the papers published as part of SPARC funding. Research articles that reference or mention these datasets, protocols, and publications were queried from NCBI (PubMed, PubMed central) repositories using the search endpoint of their Python API.12 Figure 2 shows the overall flow of data between the APIs and resources queried to get the data. The NIH RePORTER API uses project number (also known as the award number) of NIH funding associated with SPARC datasets (this is provided by the author as additional metadata required when publishing a dataset) as an input to get details including a study identifier, name of the organization that received funding, country of the organization, amount of funding received and keywords of the project topic. The NCBI API uses an identifier for PubMed Central articles to retrieve information such as article name, journal name, year of publications, and authors.\n\nWe used Google’s Firebase real-time database to store all the information retrieved via the NIH RePORTER system. The data was stored in a JSON format with read access available to anyone via a dedicated URL. The data in this database was split up into four separate sections labeled Awards, Datasets, Publications, and Protocols. All the entries within this database were given a unique identifier. These identifiers were used to link the data within the database to form a relational database. The links within the data represent the citations or use of resources within other publications. All publications within the database were uniquely identified as either SPARC-funded publications or non-SPARC publications (external publications that cite SPARC datasets and publications.)\n\nThe front-end demo of the SPARClink web page uses Vue.js to create a functional prototype of the SPARClink system. An interactive force-based undirected graph visualization was created using the D3.js JavaScript library. The choice to represent the results through such a graph was motivated by the desire to show an intuitively understandable way of showing the connected nature of citations and data reuse. The website itself is hosted on Vercel as a static front end.13 On the webpage, the visualizations can be filtered by key terms or resource type to get a better understanding of the resources created using the SPARC program. A screenshot of the webpage is shown in Figure 3.\n\nThe visualizations and the results in this figure have been filtered with the vagus and cardiac keywords.\n\nTo provide some additional functionality on the front-end demo of SPARClink, we used machine learning algorithms to enhance the user experience. We called this function of the SPARClink project the Machine Learning Data Indexing Engine.\n\nWe used the Symspell algorithm present in the scikit learn package and trained it on the vocabulary built using the SPARClink database.14 We used delete-only edit candidate generation for generating different combinations of spelling errors, and used both character-level embedding and word embedding for recommending the most probable correct spelling. The output of the spell correction algorithm was used to generate sentence-level embedding and was then compared with the embeddings of different descriptions of the items in the dataset. We obtained a ranking of all the items in the dataset based on their similarity with the searched string. The top 10 were chosen to be shown on the front end.\n\nThis module was also used to generate keywords using the keyBERT pretrained model.15 It generated the top 50 keywords associated with the whole document. It also made use of the Maximal marginal relevance algorithm to pick keywords that have a higher distance among them.16 This ensures diversity among the chosen keywords.\n\nThe engine also contains algorithms that learn vector embeddings of the descriptors of the elements present in the SPARClink database. Based on these vector embeddings, the algorithms compute the similarity between the vector representation of each word in the vocabulary with the vector representing the whole dataset and find keywords that would describe the resource. A word cloud is generated based on the relevance of these results to further enhance the user experience.\n\n\nResults\n\nUsing SPARClink, researchers can aggregate all the resources created through the SPARC program and quantify their impact. The visualization created by the SPARClink system is shown in Figure 4. The nodes in the undirected graph signify a unique SPARC resource (publication, protocol, and dataset) and the edges in the graph signify the citations or references as found by SPARClink. A well-connected graph of datasets and publications were observed but a significant number of protocols were seemingly distinct from the rest of the resources despite being pulled from the SPARC protocols.io group. This could be associated with protocols that are published on protocols.io but for which the associated datasets have not been made public yet.\n\nThe word map generated from the main dataset visualizations is shown in Figure 5. The size of the word with respect to its neighbors corresponds to the frequency and significance of the word within all the searchable metadata that we have indexed. Selecting any of the words in this map will automatically filter the SPARClink visualizations. Using a keyword filter on the graph will also prompt the top-ranking items for the keyword to be displayed on the side of the page. This ranking is shown as a scrollable list, as seen in Figure 6. Both the word map and top-ranked recommendations are continuously updating themselves when new input terms are entered via the SPARClink webpage.\n\n\nDiscussion and conclusions\n\nUsing FAIR standards can greatly improve the use of data across multiple disciplines and potentially lead to new and exciting discoveries in the field of biomedical science. The benefits of employing the FAIR data principles for data generation, curation, and sharing can, however, be hard to quantify for researchers or members of the general public. Using a system like SPARClink, researchers at all levels can get up-to-date feedback on the use of their data and all the advantages that the FAIR standards provide to efforts in advancing biomedical science. In this work, we developed such a tool for the SPARC program to enable quantification of the reuse of the FAIR SPARC resources (datasets, manuscripts, protocols).\n\nThe primary challenge in accomplishing this task lies in the fact that the SPARC datasets and protocols are not referenced in the bibliography of research manuscripts, which is the common practice. Instead, the SPARC dataset and protocol identifiers or URLs are only mentioned in the text or under supplementary materials, which makes querying this information a challenging task. Furthermore, datasets created in the SPARC program can be embargoed for up to 12 months to allow researchers enough time to document and publish their findings. However, protocols are made public immediately since protocols.io does not have an option to embargo the open publishing of these protocols. This could also add to the sparse graphs and we can expect the connectedness of this graph to improve as time goes on.\n\nIn the future, we plan on adding the Google Scholar system as an additional resource for data extraction. This should improve the connectedness of our extracted data network as well. Additional filtering functions and performance improvements for very large numbers of nodes are also planned. Currently, the tool is hosted on an independent webpage, but we also aim to integrate it directly within the SPARC portal so that visitors can conveniently visualize the reuse and impact of the different SPARC-generated resources.\n\n\nData availability\n\nAt the time of publication, the SPARClink system visualizations can be found at https://sparclink.vercel.app and are expected to be always online going forward. The backend system that queries all the publications is currently paused due to a lack of system resources. The code for SPARClink has been developed to be accessible to anyone who wants to fork the repository from GitHub and run a local version of this project. Instructions on how to run the modules locally are also available in the GitHub repository. The database of currently extracted citation data can be queried via REST protocols using the links provided below. The machine learning data indexing engine is hosted on a web server provided by pythonanywhere.com and is publicly accessible via its API endpoints. This module is also available to be run in local configuration seamlessly.\n\n\nSoftware availability\n\nSource code available from: https://github.com/fairdataihub/SPARClink\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.5550844\n\nLicense: MIT\n\n\nAuthor endorsement\n\nDavid Nickerson confirms that the author has an appropriate level of expertise to conduct this research and confirms that the submission is of an acceptable scientific standard. David Nickerson declares they were an organizer of the Hackathon in which the work described in this paper was performed. Affiliation: Auckland Bioengineering Institute, University of Auckland, New Zealand.", "appendix": "Acknowledgments\n\nWe would like to thank the NIH Common Fund’s SPARC Program and the organizers of the 2021 SPARC FAIR Codeathon for their support during the development of this project.\n\n\nReferences\n\nNational Institutes of Health: Stimulating Peripheral Activity to Relieve Conditions (SPARC).2014 [cited 2021 Oct 22]. Reference Source\n\nNational Institutes of Health: SPARC Portal.[cited 2021 Oct 22]. Reference Source\n\nQuey R, Schiefer MA, Kiran A, et al.: KnowMore: An Automated Knowledge Discovery Tool for the FAIR SPARC Datasets. bioRxiv. 2021 [cited 2021 Oct 22]; p. 2021.08.08.455581. Publisher Full Text\n\nBandrowski A, Grethe JS, Pilko A, et al.: SPARC Data Structure: Rationale and Design of a FAIR Standard for Biomedical Research Data. bioRxiv. 2021 [cited 2021 Oct 22]; p. 2021.02.10.430563. Publisher Full Text\n\nPatel B, Srivastava H, Aghasafari P, et al.: SPARC: SODA, an interactive software for curating SPARC datasets. FASEB J. 2020 Apr; 34(S1): 1–1. Publisher Full Text\n\nOsanlouy M, Bandrowski A, de Bono B , et al.: The SPARC DRC: Building a Resource for the Autonomic Nervous System Community. Front Physiol. 2021 Jun 24; 12: 693735. PubMed Abstract \| Publisher Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016 Mar 15; 3: 160018. PubMed Abstract \| Publisher Full Text\n\nThe University of Pennsylvania: Pennsieve Discover.[cited 2021 Oct 22]. Reference Source\n\nSPARC: 2021 SPARC FAIR Codeathon. SPARC Portal.[cited 2021 Oct 22]. Reference Source\n\nThe University of Pennsylvania: Pennsieve API.[cited 2021 Oct 22]. Reference Source\n\nProtocols I: Protocols.io for developers.[cited 2021 Oct 22]. Reference Source\n\nNational Institutes of Health: NIH RePORTER API.[cited 2021 Oct 22]. Reference Source\n\nSoundarajan S: SPARClink Portal.2021 [cited 2021 Oct 22]. Reference Source\n\nGarbe W: SymSpell: SymSpell: 1 million times faster spelling correction & fuzzy search through Symmetric Delete spelling correction algorithm. Github; [cited 2021 Oct 22]. Reference Source\n\nGrootendorst M: KeyBERT: Minimal keyword extraction with BERT. Github; [cited 2021 Oct 22]. Reference Source\n\nCarbinell J, Goldstein J: The Use of MMR, Diversity-Based Reranking for Reordering Documents and Producing Summaries. ACM SIGIR Forum; 2017; 51. : 209–210. Publisher Full Text" }	[ { "id": "140292", "date": "27 Jun 2022", "name": "Tao Zeng", "expertise": [ "Reviewer Expertise Machine learning and bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the paper “SPARClink: an interactive tool to visualize the impact of the SPARC program”, authors aim to introduce a web tool SPARClink for visualizing the impact of SPARC, whose methods and outcomes support FAIR guidelines.\nSPARClink should be a useful tool/software for supporting SPARC program and corresponding consortium. For the work and introduction in this paper, I have several suggestions:\nSPARC program employs FAIR, so, it is necessary to introduce more about corresponding function of SPARClink serving each FAIR guideline, e.g. for “Reusable”, what data or protocol can be reused and how other researchers can obtain and reuse them.\n\nSimilar to other programs, the data produced in SPARC would have raw data, pre-processed data, or analyzed data, or summary data, etc. The organization or level of SPARC data is better to be clearly introduced, and supply detailed cases of how SPARClink can manage and share these data.\n\nIn current implementation, “interactive force-based undirected graph visualization” is simple, and the directed relation is better to consider, e.g. the paper and its public data, and the paper and its reused data, would have different relation directions. Also, in the abstract, the authors stated “a novel knowledge graph-based structure was created to visualize …” Thus, the novelty of the network structure and representation should have highlights in revision.\n\nFor interactive visualization shown in Figure 4, or used in current web tool, the network has less information - it is better to directly show the information about each node or edge on network in web page in a scalable manner, especially for large knowledge network.\n\nThe word maps shown in Figure 5 would be a general function in many other tools, it is necessary to offer the interesting word or sentence association between key words and SPARC outcomes.\n\nIndeed, for FAIR, the software SPARClink itself is suggested to supply Docker version, so other users can easily reuse such useful framework into their applications.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "160964", "date": "26 Jan 2023", "name": "Karim Fouad", "expertise": [ "Reviewer Expertise Neuroplasticity", "data sharing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 succinct article introducing a web tool for querying and visualizing the scientific output of the SPARC program and how it has been utilized by the research community. The process begins with extracting and storing related data via APIs. Then word clouds and key word searches allow users to refine their search to then visualize the relation between the SPARC output products. The product is a very useful tool to explore the ongoing impact and use of SPARC related research and products. Potentially the impact of the tool is not really on what the authors mention (i.e., show the impact of FAIR data sharing on the overall scientific community) and thus somewhat overstated. The visualization tool simply provides a summary of the reuse of SPARC related work products, but not a comparison to other approaches for data sharing that would allow for assessing the impact of FAIR. The strength lies in the demonstration of data reuse, specific impact of their data sets, etc. This is a highly valuable tool on so many levels including strategies for future research and general education of the value of data sharing.\nThe manuscript would benefit from a few clarifications and details especially in the figure legends. For example, the difference between a SPARC publication and a data set should be explained. Are SPARC data sets not published?\nFigure 3 show a lot of white space, and the legend would benefit from more detail. What does define the size of the nodes in the visualization? Did the authors consider directional links between the items in the form of a directed graph, which would simplify comprehension of what is shown? For example, the edge of the graph could show the direction from the node citing to the one receptor of a citation.\nFigure 4 requires a legend for the different colors, an explanation for the different size of the nodes, and once again would benefit from directional edges. Lastly, not surprisingly the overwhelming links to publications mask the reuse of data sets. Maybe a different filter could be applied to show that important relation.\nMinor suggestions:\nIn the Abstract, consider adding ‘electrical’ in front of neuromodulation.\n\n3d paragraph in introduction, consider not to refer (i.e., these tools) when starting a new paragraph.\n\nPlease define “citations of SPARC-funded resources”.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "160993", "date": "16 Feb 2023", "name": "Angela Pinot de Moira", "expertise": [ "Reviewer Expertise Epidemiology", "FAIR principles" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 clearly written paper describing SPARClink, a web tool that queries all external publications and creates a database and visualisations of publications that have utilised SPARC-funded resources, including datasets, publications and protocols. As well as documenting the impact of the SPARC consortium, the tool will be an invaluable resource for researchers utilising SPARC outputs for their research.\nMy main comment to the paper is regarding the aim to demonstrate the benefits of the FAIR principles. Currently, the paper mainly focuses on the retrieval of any publication utilising SPARC resources, i.e. including publications directly funded by SPARC. To demonstrate how the tool can be used to highlight/quantify the benefits of the FAIR principles, it would be useful to provide examples of this in the paper, i.e. demonstrate the reuse of SPARC data. If I have understood correctly, this is possible by filtering extracted publications on whether they are SPARC-funded or non-SPARC funded. It would be useful to provide a visualization with the non-SPARC funded filter applied, so that the extent of data reuse can be seen.\nMy second suggestion is to include a box of key terminology in the paper. There are a lot of terms that may not be understood by the reader and although links to definitions are provided, a glossary box may aid reading.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-124
https://f1000research.com/articles/10-1201/v1	25 Nov 21	{ "type": "Clinical Practice Article", "title": "A case series of ocular involvement in bullous pemphigoid: clinical features, management, and outcomes", "authors": [ "Anahita Kate", "Swapna Shanbhag", "Pragnya Rao Donthineni", "Sayan Basu", "Anahita Kate", "Swapna Shanbhag", "Pragnya Rao Donthineni" ], "abstract": "Ocular involvement in cases of bullous pemphigoid is rare and when present, the signs are usually subtle and in the form of fine tarsal scarring and dry eye disease. The current report aims to describe the clinical features and management protocols in a series of cases with aggressive ocular manifestations at presentation. All cases of bullous pemphigoid seen between 2017 and 2020 were included in this retrospective case series. Data regarding the clinical features, treatment administered, and outcomes was collected. Five cases (n=10 eyes) of bullous pemphigoid disease with ocular involvement were included. All eyes had significant cicatricial conjunctival changes in the form of symblephara, inferior forniceal shortening, and tarsal conjunctival scarring. Conjunctival granulomas were present in 3/10 eyes. Corneal involvement in the form of punctate keratitis was present in all eyes while 4/10 eyes had an epithelial defect as well. The management of these cases involved topical therapy with corticosteroids and lubricants (n=10 eyes) while pulse doses of intravenous methyl prednisolone were administered in 5/5 cases. Pulse intravenous cyclophosphamide was supplemented in 2/5 cases. Adequate control of the disease was noted in 3/5 cases while one case had a recalcitrant form of the disease and developed a dermalised ocular surface in both eyes. The last patient was lost to follow up during the course of therapy. Bullous pemphigoid can present with an aggressive form of cicatrizing conjunctivitis similar to other variants of autoimmune blistering disorders and must be considered as a differential in cases presenting with ocular cicatricial disease. Long-term intensive immunosuppression is required for the management of these cases to preserve the visual function and the integrity of the globe.", "keywords": [ "Bullous pemphigoid", "cicatrizing conjunctivitis", "autoimmune blistering disorders", "conjunctival granuloma", "case series" ], "content": "Introduction\n\nAutoimmune blistering diseases (AIBD) are a group of disorders where the autoantibodies target antigens within the basement membrane zone (BMZ) of the skin and cause subepidermal blisters.1,2 Bullous pemphigoid (BP) is the most common type of AIBD accounting for around 80% of these blistering entities.1,2 The disease usually affects the elderly population (>60 years) and presents with tense, pruritic bullae that affect the flexor regions of the body.1,3,4 Direct immunofluorescence (DIF) of skin biopsy samples, shows a linear deposition of C3 and IgG in the BMZ.1,3 On indirect immunofluorescence, antibodies against the hemidesmosomal BP antigens 180 (BPAG2) and 230 (BPAG1) are detected in BP.1,3 These findings in the context of set clinical criteria help confirm the diagnosis of BP.5 The treatment involves long term systemic corticosteroids and non-steroidal agents, and the disease usually has a chronic course with frequent relapses especially on discontinuing the immunosuppressive medications.\n\nIn general, mucosal and ocular involvement is rare in BP and its absence is one of the clinical criteria for diagnosis.6 Literature on ocular manifestations of BP is sparse and limited to mild conjunctival scarring, dry eye disease and rarely corneal epithelial issues.7–9 The current case series aims to describe the demographic details, clinical features, treatment undergone and outcomes of five cases of bullous pemphigoid, which presented with an aggressive form of chronic cicatricial conjunctivitis.\n\n\nCase series\n\nA retrospective review of cases with bullous pemphigoid seen between 2017 and 2020 was carried out. Written informed consent for publication and use of their records was obtained from all the patients. Data regarding the baseline demographics, duration of disease prior to presentation and the details of ongoing systemic therapy was collected. All cases underwent a detailed ocular examination that included assessment of visual acuity, slit lamp biomicroscopic examination of the lids, conjunctiva, cornea, and the rest of the anterior segment. This was followed by examination of ocular surface after staining with 2% fluorescein to document the presence of disruptions in the same. A detailed evaluation of the posterior segment was also carried out. Additionally, data pertaining to the treatment initiated at our institute along with its outcomes was collected.\n\nA total of five cases with bullous pemphigoid were included in the current study. The baseline demographic details, details of systemic disorder, and full treatment details have been presented in Table 1.\n\n\n\n▪ Prednisolone acetate 1%, 8 t/d\n\n▪ Sodium hyaluronate 0.1%, 5 t/d\n\n\n\n▪ Prednisolone 10 mg OD\n\n\n\n▪ Prednisolone acetate 1%,8 t/d\n\n▪ Chloramphenicol 1% + Hydrocortisone 0.5% ointment OD\n\n▪ Hydroxypropyl methylcellulose 0.3%, 8 t/d\n\n\n\n▪ Prednisolone 10 mg OD\n\n\n\n▪ Mycophenolate mofetil 500 mg BD\n\n▪ (Day 90-ULFU)\n\n\n\n▪ Prednisolone acetate 1%, 6 t/d\n\n▪ Chloramphenicol + Polymyxin + Dexamethasone + Sodium Phosphate + Phenyl mercuric nitrate ointment BD\n\n▪ Carboxymethylcellulose 0.5%, 8 t/d\n\n\n\n▪ Prednisolone 20 mg OD\n\n\n\n▪ Cyclophosphamide 50 mg OD\n\n\n\n▪ Azathioprine 50 mg OD (Day 46-ULFU)\n\n\n\n▪ Prednisolone acetate 1% 8 t/d\n\n▪ Chloramphenicol + Polymyxin + Dexamethasone + Sodium Phosphate + Phenyl mercuric nitrate ointment TID\n\n\n\n▪ Prednisolone acetate 1% 10 t/d\n\n▪ Chloramphenicol + Polymyxin + Dexamethasone + Sodium Phosphate + Phenyl mercuric nitrate ointment TID\n\n▪ Hydropropylmethyl cellulous 0.3% gel TID\n\n\n\n▪ Prednisolone 60 mg OD\n\n\n\n▪ Cyclophosphamide 50 mg\n\n# Prednisolone acetate was tapered slowly in all cases over 6-8 weeks.\n\n* The number of surgeries is mentioned if performed more than once.\n\nA 57-year-old Asian-Indian man who was a businessman by profession, presented to our clinic with complaints of gradual diminution of vision with ocular pain and photophobia. He had developed skin blisters six months prior to presentation and had undergone a skin biopsy for this condition, which confirmed the diagnosis of BP. Although the patient was started on oral immunosuppressive medications (the details of which were not available) he was not compliant with the regimen. At presentation, the uncorrected visual acuity was 20/800 and 20/200 in the right and left eye, respectively. Details of ocular examination have been presented in Table 2. Slit lamp examination revealed significant conjunctival congestion with cicatricial changes in both eyes in the form of symblephara and shortening of the inferior fornices. These fibrotic changes were more pronounced in the lower bulbar and palpebral conjunctiva (Figure 1). On eversion of the upper lids, there was active granulation tissue in both eyes. The cornea in both eyes showed superficial punctate keratitis in the central and inferior areas with filamentary keratitis.\n\n(A-C) Slit lamp images of the right eye showing conjunctival congestion, with symblephara laterally, inferiorly and a macular scar in the inferior cornea. Inferior forniceal shortening is also noted. (D, E) Fluorescein-stained images of the right and left eyes respectively showing punctate keratitis with filaments. (F-H) Slit lamp images of the left eye showing conjunctival congestion, with exuberant granulation tissue and cicatricial changes which are more advanced than those of the right eye.\n\nThe patient was started on topical therapy along with a maintenance dose of oral steroids (10 mg/day) (full information in Table 1, Figure 2). He also received three doses of intravenous methyl prednisolone (IVMP) (500 mg), given every three weeks, following which the patient was symptomatically better with improvement in surface inflammation and decrease in the granulation tissue. Five months after presentation, the patient underwent cataract surgery with an intraocular lens as there were no episodes of disease activity in the intervening period. A pulse dose IVMP (500 mg) and cyclophosphamide (500 mg) was given prior to the surgery. Postoperatively, the patient was followed up for one year with no disease reactivation noted and vision improving to 20/60. The patient has been planned for cataract surgery in the left eye in the near future.\n\nBE: both eyes, RE: right eye, LE: left eye, IV: intravenous, MP: methyl prednisolone, CP: cyclophosphamide, TPG: tenons patch graft, AMT: amniotic membrane transplantation, SLET: simple limbal epithelial transplant, LSCD: limbal stem cell deficiency, KPro: keratoprosthesis. The numbers in brackets indicate the number of doses, if more than one was given.\n\nA 36-year-old Asian-Indian man employed in private service, presented with pain, redness, and photophobia in both eyes for six months. He was a diagnosed case of bullous pemphigoid disease based on the results of a skin biopsy and had been prescribed oral prednisolone (10 mg/day). At presentation the corrected visual acuity in the right and left eyes was 20/25 and 20/20, respectively. The inferior conjunctiva showed significant cicatricial changes, while the eversion of the upper lid revealed conjunctival granulomas, which were larger in the right eye (Table 2, Figure 3). The patient was given a pulse dose of IVMP (500 mg) along with topical therapy (full details in Table 1). Oral steroids (prednisolone 10 mg/day) were continued, and the patient was closely followed up.\n\n(A-C) Slit lamp images of the right eye showing conjunctival congestion, with two conjunctival granulomas superiorly (yellow stars) and symblephara inferiorly. (D, E) Fluorescein-stained images of the right and left eyes respectively showing punctate keratitis in the right eye. (F-H) Slit lamp images of the left eye showing conjunctival granulomas (yellow stars), with a symblepharon laterally.19\n\nA significant improvement in the surface inflammation with reduction in the size of the granulomas was noted after 1 week with the topical and systemic immunosuppressants (Figure 4). As the topical steroids were tapered, tacrolimus 0.03% eye ointment and cyclosporine 0.05% eye drops were added to the regimen. The patient continued to receive pulse IVMP (500 mg) at every follow up visit which were monthly for three months and two monthly thereafter. Oral mycophenolate mofetil was supplemented three months after presentation. No further disease exacerbation was noted and at the last follow up visit, 23 months after the initial presentation (Figure 2) the granulomas had resolved, and the eyes had no surface inflammation. The patient maintained good vision, with a corrected visual acuity of 20/, 20/20 in the right and left eye respectively. Both eyes also had partial limbal stem cell deficiency (LSCD).\n\n(A-C) Slit lamp images of the right eye showing decrease in congestion with decreased size of the granulomas (yellow stars). (D, E) Fluorescein stained images of the right and left eyes respectively showing a stable surface with no corneal staining. (F-H) Images of the left eye showing decreased inflammation and smaller granulomas (yellow stars).\n\nA 66-year-old Asian-Indian woman who was a homemaker, had complaints of redness and pain in both eyes for two months. She had oral mucosal ulcers and ruptured blisters over the upper thighs and forearms during the same period (Figure 5). The patient had previously consulted a dermatologist and was suspected to have bullous pemphigoid disease. However, the skin biopsy was not conclusive of this diagnosis. The dermatologist had started the patient on oral prednisolone, which had been tapered to 20 mg by the time of presentation to our department.\n\n(A, B) Ruptured blisters over the flexor aspect of the thighs (black arrows). (C) Oral ulcers over the hard and soft palate (yellow arrows).\n\nOn examination, the corrected visual acuity was 20/80 and 20/50 in the right and left eye, respectively. Both eyes showed conjunctival inflammation with inferior forniceal shortening and scarring in the upper tarsal conjunctiva. The right eye had two corneal defects inferiorly (Figure 6, Table 2). Schirmer’s test revealed aqueous deficiency (ADDE) in both eyes. A clinical diagnosis of bullous pemphigoid was made based on similarity with other cases. The patient was started on topical and oral immunosuppressants (full details in Table 1).\n\n(A-C) Slit lamp images of the right eye showing conjunctival congestion, with tarsal scarring superiorly (yellow arrows), limbal thickening (blue arrow) and inferior forniceal shortening. (D, E) Fluorescein-stained images of the right and left eyes respectively showing two epithelial defects in the inferior cornea in the right eye. (F-H) Slit lamp images of the left eye showing tarsal scarring (violet arrows) and inferior forniceal shortening. (I, J) Images of the right and left eye respectively after one year of follow up, showing total limbal stem cell deficiency with a pannus over the cornea. Left eye shows a dermalised ocular surface.\n\nAt the follow up visit after one month, the patient developed new skin lesions for which oral cyclophosphamide was started (Table 1, Figure 2). A sterile corneal melt was noted in the left eye at the same visit and so the patient underwent an amniotic membrane transplantation (AMT), which was repeated after one week as the original membrane disintegrated. The patient received pulse doses of IVMP peri-operatively at weekly intervals, and oral azathioprine was also added to the regimen (Table 1). The disease continued to progress despite the aggressive immunosuppression. Furthermore, the patient did not follow up regularly and one year after the initial presentation, the patient developed total LSCD with a vascularized, scarred cornea and extensive cicatricial changes (Figure 6). The left eye had a dermalised ocular surface.\n\nA 79-year-old retired Asian-Indian man, presented with complaints of pain and redness in the eyes with discharge for four months. The patient was not on any topical or systemic therapy prior to presentation. The corrected visual acuity was 20/80 and 20/100 in the right and left eye, respectively. A periocular rash with ruptured blisters was observed in both eyes (Figure 7). Ocular examination revealed thickened lid margins with distorted mucocutaneous junctions. However, no posterior migration was noted. The conjunctiva was inflamed in both eyes with intense cicatricial changes, especially in the inferior fornices, which showed shortening with symblephara. These changes were more pronounced in the left eye where the inferior fornix was obliterated. Both eyes had diffuse superficial punctate keratitis (SPKs) with a corneal epithelial defect in the left eye (Table 2, Figure 8). The patient received a pulse dose of IVMP (500 mg) with IV cyclophosphamide (500 mg) along with topical medications (full details in Table 1, Figure 2). A decrease in the size of the epithelial defect was noted after 2 months, but the surface inflammation continued to persist and so the patient received another pulse dose of IVMP and cyclophosphamide. Still, a satisfactory response was not observed and so the patient received a peribulbar injection of 1ml of 4% triamcinolone acetonide in both eyes, 3 months after the initial presentation. The patient was lost to follow up after the procedure.\n\nSome of the blisters have ruptured (yellow arrows) while the others are crusted over (red arrows).\n\n(A-C) Slit lamp images of the right eye showing thickened distorted mucocutaneous junctions, tarsal scarring superiorly and inferior forniceal shortening with symblephara. (D, E) Fluorescein-stained images of the right and left eyes respectively showing diffuse punctate keratitis in the right eye and a superior epithelial defect in the left eye. (F-H) Images of the left eye showing thickened distorted mucocutaneous junctions and obliteration of the inferior fornix. The bulbar conjunctiva also shows thickened fibrotic changes.\n\nA 27-year-old Asian-Indian man, a farmer by profession, presented with complaints of gross decrease in vision in the right eye for two weeks. He gave a history of skin blisters six months prior to presentation for which a skin biopsy was carried out and a diagnosis of bullous pemphigoid was confirmed. Systemic immunosuppressive agents were started (full details in Table 1, Figure 2). The visual acuity in the right eye was perception of light while that in the left eye was 20/50. On slit lamp examination, both eyes had significant conjunctival congestion. The right eye showed a total corneal epithelial defect with collagenolysis. The left eye had a superior corneal epithelial defect with inferior conjunctival granulomas (Figure 9). To address the issues in both eyes, the patient underwent an allogenic simple limbal epithelial transplant from a cadaveric donor, in the right eye and an AMT in the left eye. Postoperatively, the patient was started on oral steroids and cyclophosphamide (full details in Table 1). The right eye surface stabilized over two months and was completely epithelialized with a pannus formation. The left eye continued to have epithelial instability and the patient underwent a repeat AMT and a tenons patch graft for the same. Eventually the left eye developed a decompensated cornea and a keratoprosthesis (KPro) was carried out 13 months after the initial presentation. The visual acuity improved to 20/30 and the prosthesis was stable. However, one year after the surgery, extrusion of the KPro was noted and the patient underwent a repeat KPro (Figure 9). The second KPro was stable until the last follow up which was 4.5 months after the procedure. The patient had a vision of 20/100 in the left eye. No relapse of the systemic disease was seen, and the patient was on topical (prednisolone acetate 1%, tacrolimus 0.03% ointment) and systemic immunosuppression (prednisolone 10 mg and cyclosporine 50 mg).\n\n(A-C) Slit lamp images of the right eye showing congestion and chemosis with significant collagenolysis in the cornea. (D, E) Fluorescein-stained images of the right and left eyes respectively showing a total epithelial defect in the right eye and a superior epithelial defect in the left eye. (F-H) Images of the left eye showing intense fibrotic changes in the conjunctiva with two granulomas inferiorly (yellow stars). (I) Slit lamp image of the left eye with an extruded Keratoprosthesis. (J) Image of the same eye following a repeat keratoprosthesis, with a skirt amniotic membrane around the prosthesis.19\n\n\nDiscussion\n\nBullous pemphigoid disease is the most common subepidermal blistering disorder, which predominantly affects cutaneous tissue.3 Though ocular involvement is rare, a few reports of early cicatricial changes exist in literature.4,7–9 In the current series, all five cases presented with advanced disease and extensive conjunctival fibrotic changes, which were more severe in the inferior bulbar and palpebral conjunctiva. Corneal changes were also more localized to the inferior cornea suggesting a degree of exposure keratopathy, due to the inferior cicatricial changes, contributing to the disease process. The response to therapy was variable and usually correlated with the systemic response. This was seen in case 3, where the skin lesions continued to progress despite immunosuppression and a similar pattern of worsening of the ocular surface was also observed. This is in contrast to the first two cases where a good control of ocular inflammation was observed with no exacerbations of the systemic disease.\n\nAlthough BP is considered to be a disease of the elderly, 2/5 cases in the current series presented at an earlier age. A few series have described the clinical course of BP in patients younger than 60 years and have reported a more aggressive form of the disease with higher levels of circulating autoantibodies.10,11 A greater involvement of the head and neck regions has also been noted in this subgroup of population.10,11 In the present series, 4/5 cases were male, which is in contrast to the female preponderance associated with BP.4,12,13 A reversal of this trend of female predominance, with increased incidence of BP in males with advancing age has also been described.12 Ocular findings of BP described in literature include ADDE, early tarsal scarring and pannus formation.7,9 Only one case with symblephara and inferior forniceal shortening has been reported so far.7 Kiyokawa et al reported an unusual case with corneal involvement in the absence of conjunctival involvement.8 The findings in the cases described in our study represent a more aggressive form of presentation with advanced cicatricial changes and granuloma formation and have not been reported previously. The corneal involvement seen in the present series could be secondary to the inflammatory mediators over the ocular surface, the ADDE, the irregular ocular surface itself or due to the LSCD that developed over the course of the disease.\n\nSevere, progressive fibrotic conjunctival changes with concurrent LSCD and ADDE are more commonly associated with mucous membrane pemphigoid (MMP). Traditionally patients with MMP have active or scarred lesions in the oral and nasopharyngeal mucosa with occasional cutaneous involvement.6 This contrasts with BP, wherein the mucosal involvement is infrequent, and patients typically present with tense, pruritic skin blisters. Differentiating the two entities in the absence of these classical systemic findings poses a challenge because the findings on DIF for both entities are similar with linear antibody deposits in the BMZ.6,14 Therefore, in cases of isolated ocular involvement further immunopathologic workup such as DIF studies on salt-split skin, enzyme-linked immunosorbent assay (ELISA) for specific autoantibodies, etc. may be required to distinguish the two blistering disorders.6,14The clinical features of anti-epiligrin variant of MMP closely mimic those of case 3 presented in this report, can be considered a differential in such cases. The presence of antibodies against laminin 5 in patients sera detected by Western Blot and immunoprecipitation tests are used to confirm the diagnosis of this entity.15\n\nSystemic immunosuppressive therapy forms the mainstay of therapy in cases of BP, however a few cases of management with local immunosuppressive agents have also been reported.3,14 Treatment of localized cutaneous involvement with potent topical steroids such as clobetasol propionate has shown good results.1,3 A similar regimen can be adopted in isolated ocular involvement with topical or depot injections of potent corticosteroids as seen in case 4 of our series. In cases with widespread involvement administration of systemic corticosteroids is recommended. This can be given orally or in a pulsed intravenous manner.14 The advantages of the latter include a faster response rate with fewer long term side effects.14,16,17 All patients in the current series received a pulse therapy of IVMP either in isolation or in combination with cyclophosphamide. A maintenance dose of oral prednisolone was also a part of therapy in 4/5 cases. Several steroid sparing immunosuppressive agents such as azathioprine, mycophenolate mofetil, cyclophosphamide have been studied in BP, and have comparable response rates.18-20 These agents are usually added when adequate response with the maintenance steroid dose is not observed or when an increased need for pulse doses is required. Other modalities of therapy that have been used in the management of BP include dapsone, tetracyclines, biologics, immunoglobulins, plasmapheresis, etc. and these maybe effective in recalcitrant cases.1,14\n\nIn conclusion, ocular involvement in cases of BP is rare and usually subtle. In the current study, the patients presented with an advanced form of the disease characterized by severe cicatricial and granulomatous changes of the conjunctiva with LSCD, ADDE and corneal epithelial instability. Diagnosis of BP is based on a combination of classical skin blisters and a confirmatory immunofluorescence on skin biopsy. In the absence of these cutaneous findings, MMP is a close differential and additional investigations maybe required to differentiate the two diseases. Long term immunosuppression is required in the management of BP and the use of pulse IVMP with systemic steroid sparing immunosuppressive agents is usually associated with good response rates. Aggressive immunosuppression is required in cases that present with advanced ocular findings in order to preserve the visual function and to retard the chronic sequelae.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data is required.\n\n\nConsent\n\nWritten informed consent was obtained from all the patients for publication of this case report and accompanying images.", "appendix": "References\n\nDaniel BS, Murrell DF: Review of autoimmune blistering diseases: the Pemphigoid diseases. J Eur Acad Dermatol Venereol. 2019; 33: 1685–1694. PubMed Abstract \| Publisher Full Text\n\nDi Zenzo G, Marazza G, Borradori L: Bullous pemphigoid: physiopathology, clinical features and management. Adv Dermatol. 2007; 23: 257–288. PubMed Abstract \| Publisher Full Text\n\nBernard P, Antonicelli F: Bullous Pemphigoid: A Review of its Diagnosis, Associations and Treatment. Am J Clin Dermatol. 2017; 18: 513–528. PubMed Abstract \| Publisher Full Text\n\nEkong AS, Foster CS, Roque MR: Eye involvement in autoimmune blistering diseases. Clin Dermatol. 2001; 19: 742–749. PubMed Abstract \| Publisher Full Text\n\nVaillant L, Bernard P, Joly P, et al.: Evaluation of clinical criteria for diagnosis of bullous pemphigoid. French Bullous Study Group. Arch Dermatol. 1998; 134: 1075–1080. PubMed Abstract \| Publisher Full Text\n\nXu H-H, Werth VP, Parisi E, et al.: Mucous Membrane Pemphigoid. Dent Clin North Am. 2013; 57: 611–630. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVenning VA, Frith PA, Bron AJ, et al.: Mucosal involvement in bullous and cicatricial pemphigoid. A clinical and immunopathological study. Br J Dermatol. 1988; 118: 7–15. Publisher Full Text\n\nKiyokawa C, Fujito S, Mori O, et al.: Bullous pemphigoid showing unusual ocular changes. Br J Dermatol. 1998; 139: 693–696. Publisher Full Text\n\nTan JCK, Tat LT, Francis KB, et al.: Prospective study of ocular manifestations of pemphigus and bullous pemphigoid identifies a high prevalence of dry eye syndrome. Cornea. 2015; 34: 443–448. Publisher Full Text\n\nBourdon-Lanoy E, Roujeau J-C, Joly P, et al.: Bullous pemphigoid in young patients: a retrospective study of 74 cases. Ann Dermatol Venereol. 2005; 132: 115–122. PubMed Abstract \| Publisher Full Text\n\nZanella RR, Xavier TA, Tebcherani AJ, et al.: Bullous pemphigoid in younger adults: three case reports. An Bras Dermatol. 2011; 86: 355–358. PubMed Abstract \| Publisher Full Text\n\nKridin K, Ludwig RJ: The Growing Incidence of Bullous Pemphigoid: Overview and Potential Explanations. Front Med (Lausanne). 2018; 5: 220. Publisher Full Text\n\nKulthanan K, Chularojanamontri L, Tuchinda P, et al.: Prevalence and clinical features of Thai patients with bullous pemphigoid. Asian Pac J Allergy Immunol. 2011; 29: 66–72. PubMed Abstract\n\nKhandpur S, Verma P: Bullous pemphigoid. Indian J Dermatol Venereol Leprol. 2011; 77: 450–455. Publisher Full Text\n\nNischler C, Sadler E, Lazarova Z, et al.: Ocular involvement in anti-epiligrin cicatricial pemphigoid. Eur J Ophthalmol. 2006; 16: 867–869. PubMed Abstract \| Publisher Full Text\n\nShahidi-Dadras M, Karami A, Toosy P, et al.: Pulse versus oral methylprednisolone therapy in pemphigus vulgaris. Arch Iran Med. 2007; 10: 1–6. 07101/AIM.003PubMed Abstract\n\nSacchidanand S, Hiremath NC, Natraj HV, et al.: Dexamethasone-cyclophosphamide pulse therapy for autoimmune-vesiculobullous disorders at Victoria hospital, Bangalore. Dermatol Online J. 2003; 9: 2. Publisher Full Text\n\nSticherling M, Franke A, Aberer E, et al.: An open, multicentre, randomized clinical study in patients with bullous pemphigoid comparing methylprednisolone and azathioprine with methylprednisolone and dapsone. Br J Dermatol. 2017; 177: 1299–1305. Publisher Full Text\n\nVazirani J, Donthineni P, Goel S, et al.: Chronic cicatrizing conjunctivitis. Indian J. Ophthalmol. November 2020; 68(11): 2349–2355.\n\nMeurer M: Immunosuppressive therapy for autoimmune bullous diseases. Clin Dermatol. 2012; 30: 78–83. PubMed Abstract \| Publisher Full Text" }	[ { "id": "101151", "date": "13 Jan 2022", "name": "Christine Shieh", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary of the Paper\n\nThe authors present a case series of 5 patients with advanced ocular involving bullous pemphigoid (BP), a rare and sparsely characterized entity. The goal was to describe the ocular features of these patients, treatment course, and outcomes of these patients.\nAll patients were treated with systemic immunosuppression and steroid-sparing immunomodulatory therapy. All patients but one (case 4) had at least 1 year follow-up. Case 1 & 2 are examples of advanced ocular involving patients with good clinical outcomes with systemic immunosuppressive therapy.\nCases 3-5 had poorer outcomes and required more invasive interventions. Case 3 developed corneal melt refractory to medical therapy and required two amniotic membrane transplants. Her course was also complicated by irregular clinical follow-up which likely impacted her clinical course. At 1 year, she had complete limbal stem cell deficiency.\nCase 4 had peribulbar triamcinolone administered but was lost to follow-up subsequently. The clinical outcome from this case is unknown at this time. Case 5 required an allogenic limbal transplant OD and amniotic membrane OS. While the right eye stabilized, the left eye endured a complicated course requiring repeat amniotic membrane, followed by K-Pro which was complicated by extrusion and required a repeat KPro.\nThe case series accomplished its goal of presenting a variety of advanced cases with ocular involving BP, showcasing different clinical outcomes. Given that there are few reports of ocular involvement and most describe just early cicatricial changes, this series is very valuable for clinicians in understanding and treating ocular surface disease in patients with BP.\nIs the background of the cases’ history and progression described in sufficient detail?\nYes – Each of the cases are described in good detail and well-complemented by high-quality slit lamp photos. The history and clinical course/progression were further complemented by an organized timeline (Figure 2).\nAre enough details provided of any physical examination and diagnostic tests, treatment given, and outcomes?\nYes – the authors are very thorough in describing the various examination findings for each patient. There are good quality photos for each of the patients. Table 2 organizes each of the patient’s anterior segment exam findings very clearly. As for the treatment course, Figure 2 lays out a nice timeline for each of the 5 patients showing different treatment modalities and the clinical outcome.\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis, or treatment?\nYes – They do a great job of showing how ocular BP can manifest in such advanced stages. All 5 cases of advanced ocular involvement of BP required systemic immunosuppression with steroids and then steroid-sparing immunomodulators. In their discussion, they clearly state that advanced cases like these require systemic immunosuppression long-term.\nCase 3 & 5 presented with epithelial defects and had poorer outcomes/required more invasive interventions than Cases 1 & 2. Would love to have the authors include a few sentences in the discussion section about their clinical opinion as to why Cases 3 & 5 had poorer outcomes. Presumably, this was due to the degree of limbal stem cell deficiency?\nIs the conclusion balanced and justified on the basis of the findings?\nYes – they make a well supported conclusion that patients require quick immunosuppressive therapy and long term maintenance therapy as well. Patients 1&2 were successfully treated this way, and the other patients required more invasive interventions beyond systemic medical therapy.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [ { "c_id": "7742", "date": "31 Jan 2022", "name": "Sayan Basu", "role": "Author Response", "response": "Case 3 & 5 presented with epithelial defects and had poorer outcomes/required more invasive interventions than Cases 1 & 2. Would love to have the authors include a few sentences in the discussion section about their clinical opinion as to why Cases 3 & 5 had poorer outcomes. Presumably, this was due to the degree of limbal stem cell deficiency? We thank the reviewer for their comments. The progressive disease noted in case 3 was likely due to the non-compliance to the systemic immunosuppressive therapy and the progression of the underlying systemic disease as well. In case 5 the gimbal stem cell deficiency aggravated the epithelial instability and hampered the healing process resulting in repeated episodes of epithelial breakdown. These points have been incorporated within the discussion" } ] }, { "id": "101150", "date": "24 Jan 2022", "name": "Jaime D. Martinez", "expertise": [ "Reviewer Expertise Ocular surfaces 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\nExcellent review of cases. Pemphigoid is a complex disease to treat, and the importance of ocular surface details needs to be emphasized like in this article.\n\nComments:\n1. Change the word \"non steroidal\" to \"anti-inflammatory or immunosuppression\" Introduction first paragraph.\n2. Case 2 last paragraph missing number 20 on 20/20\n3. Can you add details on how the patient responded to triamcinolone injection on case 4.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [ { "c_id": "7743", "date": "31 Jan 2022", "name": "Sayan Basu", "role": "Author Response", "response": "We would like to thank the reviewer for reading and commenting on our submission. The following comments have been addressed in the manuscript. 1. Change the word \"non steroidal\" to \"anti-inflammatory or immunosuppression\" Introduction first paragraph. The required change has been made 2.Case 2 last paragraph missing number 20 on 20/20 Thank you for pointing this out. We have corrected the typographical error. 3.Can you add details on how the patient responded to triamcinolone injection on case 4 The patient unfortunately did not follow up after the triamcinolone injection and so the response to therapy is not known" } ] } ]	1	https://f1000research.com/articles/10-1201
https://f1000research.com/articles/11-123/v1	31 Jan 22	{ "type": "Research Article", "title": "Statutory lien: pre- and post- 2016 amendments to the Malaysian National Land Code", "authors": [ "Eng Siang Tay", "Ging Yee Ling", "Kwok Whee Chung", "Ging Yee Ling", "Kwok Whee Chung" ], "abstract": "Background: A statutory lien is a non-registrable security dealing under the National Land Code (NLC) in Malaysia. Lien is usually used as a short-term collateral for a repayment of debt by the proprietor. Prior to the 2016 amendments, section 281(1) of the NLC stated that a proprietor may create a lien over his land by depositing the original title with the lender, as security for a loan; and the lender shall enter a lien-holder’s caveat under section 330(1) to protect his interests. Case law has expanded the scope of section 281 to include an equitable proprietor under the definition of “proprietor” and a third-party loan with the proprietor’s consent or authority under the definition of “loan”. The 2016 amendments removed “for a loan” in sections 281(1) and 330(1). This triggers the analysis on the purposes of lien. Methods: The research methodology adopted is doctrinal legal research. Relevant sections and case law together with the Bill and Parliament’s Hansard record are examined to find the intention of the legislature and Parliament. Results: The amendments allow the lien to be used not merely as security for a loan but also for other purposes (Explanation Note 47 to the Bill). Such deletions have created an ambiguous interpretation; and may be manipulated by fraudsters. In contrast, section 241(1) provides specific purposes such as the repayment of debt, non-debt, or payment of annuity or periodic sum, for the creation of statutory charge. Conclusions: This research is only confined to the scope of lien within the Peninsular Malaysia and not the States of Sabah and Sarawak which have their respective Sabah Land Ordinance and Sarawak Land Code. A future comparative study could be made amongst the legislations. The research output may be useful for the draftsman to clear up the ambiguity in the relevant provisions.", "keywords": [ "Lien", "title", "purpose", "for a loan", "dealing." ], "content": "Abbreviations\n\nCA: Court of Appeal\n\nCap: Chapter\n\nCLJ: Current Law Journal\n\nFC: Federal Court\n\nHC: High Court\n\nMLJ: Malayan Law Journal\n\nNLC: National Land Code\n\n\nIntroduction\n\nModern financial transactions are normally backed by security to secure the loan repayment. The common security transactions are charges, liens, mortgages, pledges, guarantees and assignments. Land is usually accepted as a security due to its immovable attributes and its permanent nature. The National Land Code (Revised–2020) (NLC)1 [12] only recognises two types of security transactions as land dealings, i.e. registrable charges2 [12] and non-registrable liens3 [12]. A non-registrable lien is preferred as a short-term collateral for loan repayment by the borrower. The NLC section 5 is silent on the definition of lien [12]. Malaysian courts have referred to the common law meaning of ‘lien’ as “a right … in one person to retain that which is rightfully and continuously in his possession belonging to another until the present and accrued claims of the persons in possession are satisfied”4 [23].\n\nIt is essential that the document of title must always be kept with the lien-holder. A lien is a mere retention of title for security, thus if the title is surrendered by the lender, the lien has no more effect [21]. Section 281(1) reads together with section 330 of the NLC to provide the way for the creation and effect of liens.\n\nBy the National Land Code (Amendment) Act 2016 [10], the phrase “for a loan” in both sections 281(1) and 330(1) of the NLC 1965 were deleted [9]. Such deletion has opened to various interpretations on the purposes of lien and may lead to the misuse for an unlawful purpose. No similar provision in other jurisdictions can be referred to for review and comparison. This paper intends to analyse the purposes (pre- and post-amendments) for the creation of NLC lien.\n\n\nMethods\n\nThis paper adopts doctrinal legal research. This method examines the intention of the legislature by referring to the pre- and post- amendments to the relevant sections from the NLC from the LawNet and CLJ Online database together with the Bill and Parliament’s Hansard record. From 4 January 2021 until 21 May 2021, only Malaysian reported and unreported cases pertaining to the purpose “security for a loan” in sections 281(1) and 330(1) of the NLC 1965 were searched from LexisAdvance by using the following search terms namely “lien” and “security for a loan”. Thereafter narrowed by the search term “section 281(1)” and timeline “from 1/1/1966 until 21/5/2021” within the results. The three authors read the relevant statutory provisions and the decided cases to identify and analyse the relevancy of the cases based on the case development and expansion of its scope of the law concerned.\n\nThe authors acknowledge receipt of the ethics approval for this project issued by the Research Ethics Committee (Multimedia University) bearing the Ethics Approval Number EA0492021 dated 25/06/2021.\n\n\nResults\n\nPreliminary results based on the search terms shows 1,153 Malaysian cases shortlisted, but after narrowed by the search terms stated above, there were only 26 relevant case reports [24].\n\nCertain essential pre-requisites must first be fulfilled before a valid lien is created. Prior to the NLC Amendment Act 2016, a statutory lien must have satisfied three elements, i.e. (a) deposit with any other person the original issue of documents of title by the proprietor5 [9] (or in the case of duplicate lease, the lessee6) [9]; (b) purpose is to create a security for a loan; and (c) entry of a lien-holder’s caveat in Form 19D.\n\nMalaysian cases have interpreted and expanded the application of these sections, especially the terms of “proprietor”, ‘lien-holder”, “deposit”, “security for a loan” and “effect of entry and non-entry of a lien-holder’s caveat”. For this paper, we shall limit the analysis on the “purposes”. Section 281(1) seems to suggest that the loan is for the proprietor or the lessee. However, Malaysian cases have expanded the scope of its purposes on two aspects, (i) the purpose of loan has been expanded to include forbearance of enforcement by the lender; and (ii) the loan can be granted to the registered proprietor, equitable proprietor or even a third party with the authority or consent of the proprietor. This is shown in (Table 1) below.\n\nAlthough the outcome of the Selangor Properties case seems similar to that of the Staghorn case, the reasoning given is much commendable despite a clear definition of ‘proprietor’ in section 5 of the NLC to mean only a ‘registered proprietor’. This may open the floodgate of equitable principles in the Torrens system which places much emphasis on ‘registration’. The purpose of creating a statutory lien is much narrower compared with the registered charge under the NLC. Under section 241(1), a charge may be created for the purpose of repayment of debt [7], non-debt or payment of any annuity [6] or other periodic sum [12].\n\n\nDiscussion\n\nThe phrase “for a loan” in sections 281(1) and 330(1) of the NLC were deleted via sections 41 and 49 of the NLC Amendment Act 2016 [10]. Such deletion is unclear as to the intention of the parliament whether to widen or narrow the scope of the security to include for purposes other than for a loan, such as to create some “rights”, or as a security for “reimbursement”, as was prescribed by the Halsbury’s Law of England. Alternatively, whether the purposes would be similar to those of a registered charge.\n\nThe Explanatory Note 40 and 47 (Huraian) in the Bill7 [18] merely says that a lien can be created as security not only for loan but for other purposes. This may suggest that any other purposes to include Islamic financing, licensed or unlicensed moneylending, or for even no purpose. If the land title is in the wrong hand for no purpose, this may lead to misuse or manipulation. As both charge and lien fall under the same Part Sixteen of the NLC, a possible analogy on the purposes can be drawn from the registered charge, i.e. for the repayment of debt, non-debt, or payment of annuity or periodic sum.\n\nThe amendments have caused the lien to lose its original characteristic where the purpose of creating a lien is for the purpose of loan and not for any other purpose. Now, the deposit of the land title can be created for purpose which may not be related to the interest of the land, for example the lawyer may exercise his common law right of lien over his client’s land title in the event that the client fails to pay the professional fees to the lawyer. Instances such as the possession of the land title may be obtained through fraud or misrepresentation, or the act of deposit is done without the consent and authorisation of the proprietor may likely to happen. In Potensi Bernas Sdn. Bhd. v Lok Khi On [17], the plaintiff contended that the 1st defendant was never authorised by them to deal with the 2nd defendant in creating a lien as a security. Such amendment has also departed from the spirit of the Torrens System where it invites malpractice and thus hugely undermines the security that the NLC seeks to protect.\n\nDifferent outcomes may be reached like in Minang Bina Sdn. Bhd. v Yahya bin Mohd Said & Lain-lain [8], a lien for other purposes like safekeeping or to facilitate the subdivision of a piece of land may be allowed. “Security for share trading” as a loan under section 281(1) of the NLC which was not recognised in Citra Tani Sdn. Bhd. v Public Bank Berhad [1] may also be given due recognition. A third-party loan can be recognised without resorting to the principle in the Staghorn case [3].\n\nThe lien security need not necessarily be for a loan but for any other purpose. For instance, a father may deposit the land title to a society which would sponsor his daughter for tertiary studies. In return, the daughter has to serve a bond with the society upon graduation, failing which the society has a right to apply for an order for sale of the land. The title is deposited not for a loan to enable the daughter to further studies, but as security for the sponsorship that will be given to the daughter who in turn will provide services to the society upon her graduation. Thus, a lien is created to secure sponsorship not to the registered proprietor (father) but a third party, the daughter. The deletion of the words “for a loan” gives rise to the possibility of securing something other than a loan by using lien, and that extends to the security for a third party as well. The amendment has arguably widened the scope of purposes of lien.\n\nInitially, the same word “for a loan” in Form 19D (Entry of a lien-holder’s caveat) of the First Schedule to the NLC was not deleted following the amendment made to section 330. This omission had since been remedied by the passing of the National Land Code (Amendment of First and Fifth Schedules) Order 20178 [10].\n\nThere is an underlying danger behind the not-so-well defined scope of the purposes of lien. This may lead to a manipulation of the lien. A case to note is Lee Sean Ming & Ors v Pang Goh Kiong & Anor [4] which concerns sale and purchase agreements entered into between plaintiffs and defendants with regard to land transactions, but in reality, were sham agreements to cloak an illegal money lending transaction between the parties. The court held that these sham sale and purchase agreements were loan agreement by looking at the lien-holder’s caveat entered by the defendants on the lands.\n\n\nConclusions\n\nThe amendments seem to enlarge the scope of a lien which make it a very attractive option for the loan provider. This makes the difference between a statutory lien and a legal charge to be more distinct as the former can be used as security for some other purposes not necessarily for a loan, while the latter can only be used as security for a debt or other purposes as specified under section 241(1)(aa) or (bb) of the NLC.\n\nThe purposes of lien are not confined to “for a loan” but can include any other purposes. The effect of such amendments remains double-edged as it may bring along some drawbacks, in some illegal activities or any matters that are not related to the land.\n\nThis research is only confined to the scope of lien within the Peninsular Malaysia and not the States of Sabah and Sarawak which have their respective Sabah Land Ordinance [20] and Sarawak Land Code [19]. A future comparative study could be made amongst the legislations.\n\nIn short, the amendments brought by the NLC Amendment Act 2016 with regards to lien are meant to make people’s life easier as there will always be instances where security is needed for something other than for a loan. It is perhaps too early at this stage to comment on whether the amendments are effective or not. Whatever the outcome is, the law can always be changed for the betterment of life. Perhaps the draftsman could clear up the ambiguity in the relevant provisions.\n\n\nData availability\n\nThe dataset is available in figshare DOI https://doi.org/10.6084/m9.figshare.16411290.v1 [24].\n\nThis project contains the following underlying data:\n\n1. Aaron Walder – Halsbury Law of England (Vol.68 2021)\n\n2. Citra Tani Sdn Bhd v Public Bank Berhad (CA)\n\n3. Hong Leong Bank Bhd v Staghorn Sdn Bhd (FC)\n\n4. Heap Huat Rbber Sdn Bhd v United Overseas Bank Ltd (Ltd)\n\n5. Judith Sihombing – National Land Code Commentary\n\n6. Lee Sean Ming & Ors v Pang Goh Kiong & Ors (HC)\n\n7. Mahmud bin Hassan & Anor v CIMB Bank Bhd (HC)\n\n8. Majlis Perbandaran Pulau Pinang v Tropiland Sdn Bhd (HC)\n\n9. Mayland Lending Sdn Bhd v Rossmaizati bt Mohamad (HC)\n\n10. Minang Bina Sdn Bhd v Yahya bi Mohd Said (HC)\n\n11. National Land Code 1965 (Act 56 of 1965) (before 2016 amendments)\n\n12. National Land Code (Amendment Act) 2016)\n\n13. National Land Code (Amendment of First and Fifth Schedules) Order 2017\n\n14. National Land Code (Revised 2020)\n\n15. ORMORM Manikavasagam Chetty v TJ McGregor (HC)\n\n16. Paramoo v Zeno Ltd (FC)\n\n17. Perwira Affin Ban Bhd v Selangor Proprieties Sdn Bhd\n\n18. Perwira Habib Bank (M) Bhd v Loo & Sons Realty Sdn Bhd\n\n19. Potensi Bernas Sdn Bhd v Lok Khi On (HC)\n\n20. Rang Undang-Undang Kanun Tanah Negara (Pindaan) 2016\n\n21. Sabah Land Ordinance (Cap.68)\n\n22. Sarawak Land Code (Cap.81)\n\n23. United Overseas Bank Sdn Bhd v UJA Sdn Bhd\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to thank the Multimedia University’s Siti Hasmah Digital Library for accessibility of the online databases.\n\n\nReferences\n\nCitra Tani Sdn. Bhd. v Public Bank Berhad [2020] MLJU 201(CA).\n\nHeap Huat Rubber Co. Sdn. Bhd. v United Overseas Bank Ltd. [1992] 3 CLJ 1589 (HC).\n\nHong Leong Bank Bhd. (formerly known as Hong Leong Finance Bhd.) v Staghorn Sdn. Bhd. & Other Appeals. [2008] 2 MLJ 622 (FC).\n\nLee Sean Ming & Ors v Pang Goh Kiong & Anor. [2014] 1 LNS 1780 (HC).\n\nMahmud bin Hassan & Anor v CIMB Bank Bhd. [2018] 8 MLJ 663 (HC).\n\nMajlis Perbandaran Pulau Pinang v Tropiland Sdn. Bhd. [2003] 6 MLJ 387 (HC).\n\nMayland Lending Sdn. Bhd. v Rossmaizati bt Mohamad & Anor. [2015] 7 MLJ 216 (HC).\n\nMinang Bina Sdn. Bhd. v Yahya bin Mohd Said & Lain-lain [1991] 1 CLJ 70 (HC).\n\nNational Land Code 1965 (Act 56 of 1965), section 5, Part Sixteen, and sections 241(1)(aa) & (bb), 281(1), 330(1) before 2016 amendments.\n\nNational Land Code (Amendment) Act 2016 (Act A1516), (w.e.f. 1 January 2017 in general except some sections), sections 41 & 49 and P.U.(B) 527/2016 & P.U.(B) 531/2016.\n\nNational Land Code (Amendment of First and Fifth Schedules) Order 2017 (P.U. (A) 247/2017).\n\nNational Land Code (Revised – 2020) (Act 828), section 5, Part Sixteen, and sections 241(1)(aa) & (bb), 281(1), 330(1).\n\nORMORM Manickavasagam Chetty v TJ McGregor [1933] MLJ 295 (HC).\n\nParamoo v Zeno Ltd. [1968] 2 MLJ 230 (FC).\n\nPerwira Affin Bank Bhd. (formerly known as Perwira Habib Bank Malaysia Bhd.) v Selangor Properties Sdn. Bhd. & Ors. [2010] 3 CLJ 45 (CA).\n\nPerwira Habib Bank (M) Bhd. v Loo & Sons Realty Sdn. Bhd. & Satu lagi (No 1) [1996] 3 MLJ 409; [1996] 4 CLJ 171 (CA).\n\nPotensi Bernas Sdn. Bhd. v Lok Khi On [2020] MLJU 1004 (HC).\n\nyesRang Undang-Undang Kanun Tanah Negara (Pindaan) 2016, Huraian 40 & 47. .\n\nSabah Land Ordinance (Cap. 68).\n\nSarawak Land Code (Cap. 81).\n\nSihombing J: The National Land Code Commentary – Lien, Chapter 6 para 1375.\n\nUnited Overseas Bank (M) Sdn. Bhd. v UJA Sdn. Bhd. [2009] 6 MLK 857 (CA).\n\nWalder A: Halsbury’s Law of England – Liens. 2021; Vol.68. para 902.\n\nTay ES, Ling GY, Chung KW: Statutory Lien: Pre- and Post- 2016 Amendments to the Malaysian National Land Code. figshare. Dataset. 2021. Publisher Full Text\n\n\nFootnotes\n\n1 Act 828, with effect from 15 October 2020. This NLC has replaced the National Land Code 1965 first enacted in 1965 (Act 56 of 1965) (‘NLC 1965’). Both NLC and NLC 1965 will be used in this paper since almost all cases were decided under the NLC 1965. Furthermore, the amendments made in 2016 was based on the NLC 1965 and not the current revised NLC although the content remains the same.\n\n2 Chapter 1-5, Part Sixteen of the NLC (ss 241-280).\n\n3 Chapter 6, Part Sixteen of the NLC (s 281), read together with sections 330 & 331. See also section 206(2)(b) of the NLC.\n\n4 Walder A, Halsbury’s Law of England – Liens, Vol.68, 2021, para 902. Malaysian cases which quoted the earlier editions of the Halsbury’s Law of England (with same definition) include: ORMORM Manickavasagam Chetty v TJ McGregor [1933] MLJ 295 at 296; and Perwira Affin Bank Bhd. (formerly known as Perwira Habib Bank Malaysia Bhd.) v Selangor Properties Sdn. Bhd. & Ors [2010] 3 MLJ 43 (CA) at [12].\n\n5 Under section 5 of the NLC, the word “proprietor” means “any person or body for the time being registered as the proprietor of any alienated land”.\n\n6 Section 5 of the NLC defines “lease” to mean a registered lease or sub-lease of alienated land. The word “lessee”, although not defined, refers to any person or body for the time being a lease registered in his favour.\n\n7 See Huraian number 40 of Rang Undang-Undang Kanun Tanah Negara (Pindaan) 2016 which states that “40. Fasal 41 bertujuan untuk meminda seksyen 281 Kanun untuk membolehkan lien dibuat sebagai jaminan dan bukan semata-mata bagi tujuan pinjaman.” (in Malay). See also Huraian number 47 for section 49 of the NLC Amendment Act 2016.\n\n8 P.U. (A) 247/2017 (w.e.f. 30 August 2017). The inconsistencies arose from NLC Amendment Act 2016 has been rectified where section 2(d) of the said Order was subsequently amended by deleting the words “for a loan” in paragraph (2) of the Lien-holder’s Form in Form 19D." }	[ { "id": "121680", "date": "11 Feb 2022", "name": "Hang Wu Tang", "expertise": [ "Reviewer Expertise Land Law" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments:\nIn this article, the authors study the 2016 amendments of sections 280 and 330 of the National Land Code in relation to statutory liens. It would be helpful to set out the ambit of sections 280 and 330 of the National Land Code for the reader’s convenience at an earlier stage. The authors point out that statutory liens used to be created “for a loan”. But the recent amendments have deleted the words “for a loan”. This leads the authors to postulate that statutory liens may now be procured for other purposes. While this is found in a later part of the article, it would be helpful for the authors to set out what other purposes might be allowed or disallowed pursuant to the amendments. These include a lawyer’s common law right of a lien over his or her clients land title in relation to unpaid professional fees, security for share trading, liquidated damages (in the example of the father creating a statutory lien for the daughter’s scholarship sponsor) and loans for third parties.\nSeveral drawbacks to the widening of statutory liens were identified by the authors. The authors argue that such statutory liens may lead to circumstances where the lien is created pursuant to fraud, misrepresentation, illegality, or a situation where the landowner did not consent to the creation of the statutory lien. This line of argument needs to be developed to be sustainable. As currently drafted in the article, I am not convinced by this argument. If there is in fact of fraud, misrepresentation, illegality or non est factum, then this would be vitiating factors that will set aside the statutory lien. More intriguing is a question hinted at in this piece but not fully developed: Should the policy makers make it so easy for land to be used as security for other purposes apart from a loan? Would this give rise to the use of land in ways that might be unforeseen i.e. security for trading in shares, cryptocurrencies, etc? Would this lead to negative social implications where the landowner’s family would be dispossessed when the lien is enforced?\nFor ease of reading, I would also suggest that the discussion of the case law be expanded. The relevant case law is dealt with in one liners, and I found it difficult to follow the authors’ arguments and appreciate the significance of the cases.\nFinally, I am not sure whether the conclusion is consistent with the main flow of the argument in the paper. The tenor of the argument in the paper appears to be against the expansion of the statutory lien. Yet, in the conclusion, the authors state that the amendments were “meant to make people’s life easier as there will always be instances where security is needed for something other than for a loan”. If the authors are in fact supportive of the amendments, then the preceding sections of the article should make this clearer.\n\nThe following work and literature should be considered and cited in this article:\nWong Kim Fatt, “Twenty-Five Years of the Caveat System under the National Land Code1965” 1\n\nZeno Ltd. v. Prefabricated Construction Co. (Malaya) Ltd. & Anor (1967) 2 for the nature of the lien under the pre-cursor of the lien in the Federated Malay States\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": [] }, { "id": "123537", "date": "21 Mar 2022", "name": "Nuarrual Hilal Md Dahlan", "expertise": [ "Reviewer Expertise Land Law" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article discusses the new amendment made on lien, pursuant to the amendment made to National Land Code.\nThe discussion is simply on the issue of the ‘purposes’ of the creation of lien. Through the amendment, the purposes of lien are not just limited to ‘loan’. It will also cover other situations, i.e. non-loan transactions. This includes the contention of the authors to cover the situation of Islamic finance and banking transactions which the reviewer agrees. The authors should elaborate and explain this situation, i.e. the Islamic finance transactions that involve lien.\nExplain the Islamic finance concept and why lien is not just restricted to loan transactions. In Islamic finance and banking, the Islamic bank provides facilities to customers. The facilities are not ‘loans’, but Islamic finance facilities that involve buying and selling and/or transactions that are acceptable under Islamic Law. The purpose of Islamic Law is to prevent the practice of riba’ (usury) and other elements affronting the religion of Islam.\nFurther, the authors’ view that the creation of lien to other ‘non-loan’ transactions can cause fraud, underlying danger, or other inequitable and unjust practices is not correct. The authors premise this on presumption. The reviewer does not share the same view. Thus, on this matter, the authors should provide more elaboration and provide proof and examples of the potential fraud, underlying danger, or other inequitable and unjust practices due to the widening ‘purposes’ of creation of lien.\nThe cases provided by the authors also are too succinct and simple. Please provide the relevant facts and relate the principles to the discussion of the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-123
https://f1000research.com/articles/10-433/v1	01 Jun 21	{ "type": "Systematic Review", "title": "Quality of information in news media reports about the effects of health interventions: Systematic review and meta-analyses", "authors": [ "Matt Oxman", "Lillebeth Larun", "Giordano Pérez Gaxiola", "Dima Alsaid", "Anila Qasim", "Christopher James Rose", "Karin Bischoff", "Andrew David Oxman", "Lillebeth Larun", "Giordano Pérez Gaxiola", "Dima Alsaid", "Anila Qasim", "Christopher James Rose", "Karin Bischoff", "Andrew David Oxman" ], "abstract": "Background Many studies have assessed the quality of news reports about the effects of health interventions, but there has been no systematic review of such studies or meta-analysis of their results. We aimed to fill this gap (PROSPERO ID: CRD42018095032). Methods We included studies that used at least one explicit, prespecified and generic criterion to assess the quality of news reports in print, broadcast, or online news media, and specified the sampling frame, and the selection criteria and technique. We assessed criteria individually for inclusion in the meta-analyses, excluding inappropriate criteria and criteria with inadequately reported results. We mapped and grouped criteria to facilitate evidence synthesis. Where possible, we extracted the proportion of news reports meeting the included criterion. We performed meta-analyses using a random effects model to estimate such proportions for individual criteria and some criteria groups, and to characterise heterogeneity across studies. Results We included 44 primary studies in the qualitative summary, and 18 studies and 108 quality criteria in the meta-analyses. Many news reports gave an unbalanced and oversimplified picture of the potential consequences of interventions. A limited number mention or adequately address conflicts of interest (22%; 95% CI 7%-49%) (low certainty), alternative interventions (36%; 95% CI 26%-47%) (moderate certainty), potential harms (40%; 95% CI 23%-61%) (low certainty), or costs (18%; 95% CI 12%-28%) (moderate certainty), or quantify effects (53%; 95% CI 36%-69%) (low certainty) or report absolute effects (17%; 95% CI 4%-49%) (low certainty). Discussion There is room for improving health news, but it is logically more important to improve the public’s ability to critically appraise health information and make judgements for themselves.", "keywords": [ "news", "news media", "news reports", "health news", "systematic review", "meta-analysis", "infodemic" ], "content": "Introduction\n\nFor decades, researchers have studied and criticised the quality of health information in the mass media. In 1972, The New England Journal of Medicine—the “most prestigious” general medical journal in the world1—published an “examination” of health information on American television (TV).2 The paper ended in reproach: “The potential of television to inform, instruct and educate should not continue to be wasted and abused, but rather should be used as an integral part of a plan designed to deliver better health care.”\n\nMisleading health information in the mass media continues to be a hot topic. In 2020, the World Health Organization reported that the Coronavirus disease (COVID-19) had been accompanied by an “infodemic”: “an over-abundance of information—some accurate and some not—that makes it hard for people to find trustworthy sources and reliable guidance when they need it.”.3\n\nMany studies of health news have focused on “traditional” news media, including print, broadcast, and news websites. With the advent of social media, traditional news media have increased competition as a source of lay health information. However, people still get health information from the latter, directly or via social media. Chew and Eysenbach found that about 60% of more than 5000 tweets about the H1N1 outbreak in 2009 included links and about a quarter of those links were to news websites, the most common destination.4 In comparison, less than 5% of links were to government and public health agencies or to other social media, combined.\n\nMany of the studies focusing on news media have further focused on news reports about the effects of health interventions. This includes “modern” medicine (aka. “academic”, “conventional” or “Western” medicine); “alternative” medicine (aka. “complementary”, “traditional” or “natural” medicine); screening; surgery; devices; diet; exercise; lifestyle interventions; and health systems and policies. We were unable to find a systematic review of such studies (see extended data - S1, S2 and S3 Files104).5 We then aimed to fill this gap, providing meta-analytical estimates of the quality of news reports about the effects of health interventions, and potentially informing further and related research.\n\nThe first part of this study was submitted by the first author, MO, to the University of Oxford as his dissertation, in partial fulfilment of the requirement for the award of Master of Science (MSc) in Evidence-Based Health Care, under the title “Criteria used to measure the quality of news media report about the effects of health interventions: Systematic review” (see extended data - S4 File104).\n\nPrimary:\n\n• To assess the quality of information in news media reports about the effects of health interventions.\n\nSecondary:\n\n• To assess, map and group criteria used to measure the quality of information in news media reports about the effects of health interventions.\n\n\nMethods\n\nThis review was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (ID: CRD42018095032). The protocol was published on the Informed Health Choices project website as “Quality of news media reports about the effects and costs of health interventions: Systematic review protocol” (see extended data - S1, S2 and S3 Files104).5 Appendixes to the protocol were published in a separate compressed folder, on the same website. A Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Checklist6 has been included in the Reporting guidelines for this review.\n\nPrimary studies had to satisfy four eligibility criteria to be included in the review (Table 1): two related to the sample of news reports included in the study; one related to the outcome measure; and one related to reporting of the study. We included studies in any language in the qualitative summary, but we only included studies in English or Norwegian in the meta-analyses (the two languages spoken fluently by the primary investigator, MO). We did not place any explicit limitation on study design, but—in effect—case studies of a single news report and purely qualitative studies were ineligible.\n\nThe first eligibility criterion was that the sample include content labelled by the researchers as “news”, taken from newspapers or magazines (print); television, radio or podcasts (broadcast); or dedicated news websites (online). We placed no limits on where in the world or when the news reports were published.\n\nSecond, the sample had to include news reports about the effects of health interventions. Other types of health news include non-reports (e.g. features or opinion pieces) and other categories of news reports about health (e.g. reports about the health effects of exposures or about health-related ethical or legal issues). We defined “health intervention” as any action intended to improve the health of individuals or communities, as per the Glossary of Evaluation Terms for Informed Treatment choices (GET-IT).7 We defined “effect” as a negative or positive change or difference in a health outcome, again as per GET-IT.\n\nThird, the researchers had to have used at least one explicit, prespecified and generic criterion to measure quality, herein called a quality criterion. “Quality” was defined as how conducive the report is to informed decisions or, conversely, how misleading it is. Information about the effects of health interventions can be directly misleading—such as claims that an intervention causes an outcome when it is only associated with it—or it can be misleading by omission, such as omitting absolute effect estimates, particularly when the baseline risk is small.\n\n“Criterion” was defined as a standard of quality that could be satisfied or not. “Generic” meant that the quality criterion was not specific to an intervention (e.g. a drug), category of interventions (e.g. “modern” medicine), or condition (e.g. cancer), or specific to any evidence (e.g. findings from a trial). We excluded studies if they only measured the quality of the evidence cited in the news reports; only measured the factual accuracy of the reports; or only described the reports, as opposed to explicitly assessing their quality. We included quality criteria related to cost of the intervention.\n\nThe fourth and final eligibility criterion was that the study specified the sampling frame (where the news reports were sampled from), the selection criteria for the news reports, and the selection technique (how the reports were sampled).\n\nTo clarify the eligibility criteria, three reviewers piloted them on a sample of five studies from a list of potentially eligible studies of which we were aware before finalising the protocol (Appendix 1 of the protocol; extended data - S2 File104).5\n\nWe searched for eligible primary studies indexed in PubMed on May 24, 2018, and Google Scholar on June 21 and 22, 2018. We searched directly in Open Grey and Grey Literature Report on June 22, 2018, and in ProQuest Dissertations & Theses (Global Full text plus UK and Ireland abstracts) on June 25, 2018. We updated the PubMed search on Aug. 30, 2019.\n\nWe conducted citation searches for papers describing the development of tools for assessing the quality of news reports about health: the Index of Scientific Quality;8 the Quality Index for health-related Media Reports;9 and a checklist for improving drug information in the general press.10 We also checked the reference lists of those three papers. Finally, we conducted citation searches and reference lists checks for an arbitrary selection of eligible studies. S5 File in the extended data104 is a detailed description of the search strategy, including search strings.\n\nScreening search results for inclusion in the qualitative summary consisted of two rounds. First, two reviewers screened titles and abstracts. Second, two reviewers screened full texts of studies included after the first round. The two discussed and resolved disagreements after each round. Where two reviewers could not reach a consensus, a third was brought in to arbitrate. Screening reference lists and the results of citation searches included a third, initial round, wherein only one reviewer screened titles. We also screened all of the potentially eligible studies of which we were aware before finalising the protocol (Appendix 1 of the protocol; extended data – S2 file104). We screened search results using Rayyan.11\n\nIf a study included a mix of news reports about the effects of health interventions and other types of health news, we excluded the study from the meta-analyses unless results for the news report about the effects of health interventions were reported separately for at least one eligible quality criterion. Further, we excluded studies from the meta-analyses if they did not include any eligible quality criteria or any such criteria for which results were adequality reported (see the section “Assessing criteria”). Judgements about excluding studies from the meta-analyses were made independently by two reviewers, who then discussed and resolved disagreements.\n\nData were extracted by a single reviewer and entered into spreadsheets (Microsoft Excel, Version 16.49). We only extracted reported data. For example, if a study did not include the country or countries in which the sample of news reports were published, we did not attempt to code the individual news reports by country or contact the study authors for that information. A second reviewer checked a sample of the extracted descriptive data and checked all the extracted quality criteria that were included in the meta-analyses, as well as results for those criteria.\n\nFor the qualitative summary, we extracted descriptive data about the samples of news reports and the tools (sets of quality criteria) used to assess the reports. Data about the sample of news reports included: the categories of media in which the reports were published; the countries in which they were published; the time period in which they were published; the categories of health interventions that were the subject of the reports; and the financial models of the news outlets that published the reports. We also extracted study objectives, sampling frame, selection criteria and technique, and reported subgroup analyses.\n\nAll categories and the time periods were predetermined in the protocol. The categories of media were newspaper; magazine; radio; podcast; television; and news website. The categories of intervention were: “modern” medicine; “alternative” medicine; screening; surgery; devices; diet; exercise; lifestyle; and systems and policies. The two categories of financial model were non-commercial (public or independent) and commercial.\n\nWe extracted quality criteria verbatim. For the meta-analyses, we extracted all response options for each included quality criterion, the number of relevant news reports (reports about the effects of health intervention) to which the quality criterion was applied, and the results for those reports. Some quality criteria were not applied by researchers to the total sample of relevant reports in a given study, meaning the researchers did not consider the quality criterion universally applicable.\n\nAgain, if a study included a mix of news reports about the effects of health interventions and other types of health news, we only included results for the former. We excluded studies if those results were not reported separately.\n\nFor the meta-analyses, we were interested in the proportion of relevant news reports that satisfied a given quality criterion. If this was not reported explicitly, we imputed the proportion using reported data, if possible. We excluded quality criteria if the proportion was not explicitly reported and it was impossible to calculate the proportion based on the reported data—for example, if only mean scores were reported.\n\nAfter selecting studies to be included in the meta-analyses (Figure 1), two authors assessed criteria used in those studies: both quality criteria and other criteria, such as descriptive criteria or criteria measuring factual accuracy, using spreadsheets. We excluded inappropriate criteria, as well as criteria for which it was not possible to extract results for only news reports about the effects of health interventions.\n\nWe assessed each criterion in each study, even if the researchers used a tool used in a previous study, in case it had been modified. For example, the Media Doctor Australia tool is used in several studies, including those by Smith et al.12 and Bonevski et al.13 However, while Smith et al. asked whether there is “objective evidence to support the treatment”, Bonevski et al. asked whether the study methodology is reported, which is more specific.\n\nTwo reviewers independently assessed criteria, then discussed and resolved any differences. We excluded criteria if they were non-generic or if they assessed factual accuracy, if they were descriptive, or if they were not clear or sensible (i.e. if their face validity was inadequate14). We also excluded global scores and composite quality criteria because they can be difficult to interpret, and they are difficult to compare across studies using different scoring systems.\n\nWe mapped and grouped quality criteria, using spreadsheets, to be able to synthesise results across studies using different tools. For the mapping, we used the Informed Health Choices (IHC) Key Concepts framework. The most recent version of the framework was published on IHC website in 2019 and includes nine high-level concepts and 49 low-level concepts organised in three overarching groups (see extended data - S6 File104).15 The IHC Key Concepts are essential concepts for assessing information about the effects of health interventions and making informed choices.15–17 The framework is based on a mix of evidence and logic. It has a combination of characteristics that make it unique, as well as uniquely useful for this study. First, it has been developed systematically, transparently and iteratively. Second, it only includes concepts that are intended to be relevant to the general public (typically the target audience of health news). And third, it is intended for mapping and assessing measurement tools (amongst other purposes). Moreover, the starting point for the framework included checklists for journalists and input from journalists.18\n\nTwo reviewers independently mapped the included quality criteria against the low-level IHC Key Concepts, then discussed and resolved any disagreements. Finally, one reviewer grouped the criteria, informed by the results of the mapping, and a second reviewer assessed whether the groups were sensible (i.e. face validity14).\n\nWhere an included quality criterion had more than two response options, one reviewer dichotomised the categories and a second reviewer assessed the dichotomisation. For example, for studies that used the Index of Scientific Quality, where response options range from a score of one to five, we considered a score of four or five as satisfying the quality criterion.\n\nFurthermore, we reframed negative quality criteria as positive, and paraphrased all quality criteria, for the data to be consistently framed and worded, and for the criteria to be succinctly labelled. For example, Bubela et al. report the number of news reports that “did not” specify whether a study was randomized, which was reframed as the number of news reports that did specify whether a study was randomized.19\n\nFor each individual quality criterion and group of criteria, we estimated mean proportions with 95% confidence intervals (CIs), using a random effects model, quantifying precision using 95% confidence intervals. Where criteria groups included more than one criterion from the same study, we included results for both criteria. We performed statistical analysis using Stata 16 (StataCorp LLC, College Station, Texas, USA). Each of the included studies provided data on the number of relevant news reports assessed and how many of those reports met a given criterion. For each study and quality criterion for which necessary data were available, we estimated the proportion of relevant news reports meeting the criterion. These point estimates and 95% confidence intervals were transformed to the logit scale for meta-analysis (Stata's default scale for meta-analysis of proportions). We had anticipated substantial between-study heterogeneity and therefore used random effects meta-analyses. We estimated the percentage of variance that can be attributed to between-study heterogeneity rather than chance (I2, expressed as a percentage) and calculated P-values to test the null hypotheses of no between-study heterogeneity using Cochran's Q statistic. We back-transformed meta-analytical estimates from the logit scale to present estimates as percentages (in forest plots summarizing meta-analytical results from different criteria groups) or proportions (in forest plots for individual groups).\n\nTwo reviewers assessed whether what the different quality criteria in each group measured was similar enough that it was sensible to report an overall estimate. If a group included a single quality criterion, we did not report an overall estimate.\n\nTwo reviewers independently assessed the risk of detection bias for each criterion included in the meta-analyses, then compared judgements and resolved disagreements. Detection bias was assessed using three questions:\n\n1. Was the criterion applied by at least two researchers independently?\n\n2. Were the researchers blinded in terms of the journalist and news outlet?\n\n3. Does the criterion require substantial judgement?\n\nIf the authors did not state how many researchers applied the criterion or whether the researcher(s) did so independently, we recorded the answer to the first question as “unclear” and counted it as a “no”. If from looking at the criterion in and of itself, it was unclear how much judgement was required, we looked at whether the researchers reported or referenced any guidance for applying it. If they did, we looked at how detailed and specific the guidance was. If the researcher(s) applying the criterion received training, but the authors did not report what this entailed—i.e. what guidance was provided—we considered the criterion to require substantial judgement.\n\nWe considered the risk of detection bias as low if the criterion did not require substantial judgement. We considered the risk of detection bias as moderate if the criterion required substantial judgement, but at least two researchers applied the criterion independently and they were blinded in terms of the journalist and outlet. We considered the risk of detection bias as high if the criterion required substantial judgement and only one researcher applied it or the researchers were not blinded.\n\nIf there was a high risk of bias for criteria providing ≥50% of the weight in a given criterion group, we considered there to a be a high risk of bias for the overall estimate.\n\nWe used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach as a framework for rating the quality of evidence.20 GRADE considers five factors that can lower the certainty of evidence for effect estimates: risk of bias, inconsistency of results, indirectness of evidence, imprecision, and publication bias. We did not consider publication bias, as we are not aware of any research documenting publication bias in studies of the quality of news reports or similar studies. We also did not consider directness, which was irrelevant.\n\nAgain, if there was a high risk of bias for criteria providing ≥50% of the weight in a given group, we considered there to be a high risk of bias for the overall estimate. If a CI is wider than 0.25 (a quartile), we considered there to be important imprecision, based on guidance from the Cochrane Effective Practice and Organization of Care (EPOC) group.21 We used an I2 of >75% as a rule of thumb for there being substantial inconsistency.\n\nGRADE factors for increasing the certainty of the evidence were irrelevant.\n\nWe report the main deviations from the protocol here. In S7 File (see extended data104), we provide detailed information about all deviations. Some of the deviations were in part due to the originality of our research question in terms of a systematic review and meta-analysis, meaning there was lack of methodological guidance or precedence for the study as a whole. In other words, we were in “unchartered waters”. Other deviations were due to limited time and resources. This review had no funding and as noted in the background section, the first part of the review was MO's MSc dissertation, meaning he was required to do as much as possible of that work himself.\n\nIn terms of search strategy, we did not search Scopus as planned, aside from citation searches, since the time saved outweighed the likelihood of identifying additional eligible studies, based on the results of searching the other databases. Also, we only conducted reference list checks and citation searches for arbitrary samples of included studies, due to limited time and resources, as well as decreasing yield (extended data – S5 File104).\n\nIn terms of eligibility, we made explicit that at least one of the criteria that researchers in a given study used to assess quality had to be prespecified and generic, and used specifically to assess the quality of news reports, not describe their content or assess the quality of underlying evidence. As mentioned, we included studies in any language in the qualitative summary, but only included studies in English and Norwegian (the two languages spoken fluently by the primary investigator, MO) in the meta-analyses.\n\nIn terms of data extraction, a second reviewer checked all data extracted for the meta-analyses, but only a sample of data extracted for the qualitative summary due to limited time and resources. There were no errors in the sample. In terms of assessing risk of bias, we were originally going to assess selection bias by looking at how news reports were sampled: randomly, sequentially or other. However, we found this told us more about applicability than bias, since samples typically consisted of all news reports that met the researchers’ eligibility criteria, rather than a sample of those reports. Therefore, we only assessed the risk of detection bias, as described earlier.\n\nIn terms of synthesising results and rating the quality of the evidence, we have not reported overall estimates for groups of quality criteria if what those criteria measure is too different for a synthesis to be sensible, despite the criteria being clearly related. Nor have we reported overall estimates for groups that include a single criterion. Further, we did not rate the quality of the evidence in these cases. Where we do report an overall estimate and rate the certainty of the evidence, if we assessed the risk of bias as high for criteria providing ≥50% of the weight in a given group, we assessed the risk of bias as high for the group as a whole. We did not consider assessing risk of bias for whole criteria groups in the protocol.\n\n\nResults\n\nWe screened 2063 unique records and retrieved and assessed 140 full texts for eligibility, as shown in the PRISMA flow diagram (Figure 2). We identified 44 primary studies for inclusion in the qualitative summary (see extended data - S1 Table104): 31 in English, five in German, four in Spanish, three in Norwegian and one in Slovenian. We excluded most (78) full-text articles from the qualitative summary based on one or more of the four eligibility criteria. We excluded another 16 full-text articles that were not research (e.g. commentaries), and two duplicates: one doctoral thesis that is a composite of other studies that we assessed22 and one working paper23 superseded by the final publication.24 The third eligibility criterion—that the researcher had used at least one explicit, prespecified and generic quality criterion—was least often satisfied. We excluded 47 full-text articles solely for failing to satisfy that eligibility criterion.\n\nOf the 44 studies included in the qualitative summary, we included 18 (41%) in the meta-analyses. Of the 26 studies only included in the qualitative summary, we excluded nine (20%) from the meta-analyses for being in a language other than English or Norwegian; 16 for inadequate reporting (36%); and one (2%) where the sample of news reports25 was included in a larger sample in a later study.26 Only one of the 18 studies included in the meta-analyses was in Norwegian,27 meaning the rest were in English.\n\nSamples\n\nTable 2 is an overview of the samples of news reports in the studies included in the qualitative summary. Descriptive data for each study can be found in S2 Table in the extended data.\n\na As per categories pre-specified in the protocol: newspapers, magazines, TV, radio, podcasts, and news websites.\n\nb The same study may be counted in several categories.\n\nc Time period when news reports were published or studied, depending on what authors reported.\n\nd As per categories pre-specified in the protocol: “modern” medicine, “alternative” medicine, screening, surgery, devices, diet, exercise, lifestyle, and systems and policies.\n\ne The authors do not specify whether the news reports were sampled from newspapers, magazines or both.\n\nSample sizes varied from 17 to 3096 units. We use “units” here since some samples include content besides news reports, such as non-news websites,28 or include health news that is not news reports about the effects of health interventions, for example news features29 or news reports about the effects of exposures, such as recreational cannabis (as opposed to medical cannabis).30\n\nNewspaper was the most common medium, with newspaper content explicitly included in 35 of the 39 studies (90%) in which a medium was specified (Table 2). However, the number of studies that included newspaper content versus content from news websites should be interpreted with caution, since newspaper content may also have been published online. In the study with the second-largest sample, Walsh-Childers et al. write that news reports “must have been published on the news organization's website,” but categorise those reports only as newspaper content.26\n\nThe United States (US) was the most common country in terms of where content assessed in the included studies was published. Of the 40 studies where at least one country was specified, 12 samples (30%) included content published in the US (Table 2). Twenty-five (63%) of those 40 studies included content published in English-speaking countries (see extended data - S2 Table104).\n\nWe categorised time periods according to what authors reported. This was in some cases the period in which news reports were published and in others the period in which reports were assessed. Differences between the two are likely minor, since all studies focused on the recent past (relative to the time that the study was conducted). The earliest year of publication was 1996.31\n\n“Modern” medicine was included in 24 of 32 samples (75%) where a category of interventions is specified. In some cases, it was not possible to use the categories pre-specified in the protocol for this review. For example, Haneef et al. included news reports about “non-pharmacological” interventions,32 which could include “Alternative” medicine, devices, diet, etc. In these cases, we used the category “Other”.\n\nFinancial model was not specified in any of the included studies—i.e. it was not explicitly reported whether the news outlets that published news reports included in the sample were commercial or non-commercial (public or independent).\n\nTools\n\nThe tools used to assess the quality of news reports in each study are presented with the descriptive data in S2 Table in the extended data.104 Most studies used a tool that was, to varying degrees, original or modified. Only one tool was used in >4 of the 44 studies included in the qualitative summary: the Index of Scientific Quality, which was used in seven studies (16%).28,30,31,33–36 However, even more studies used tools that were to some extent, directly or indirectly, based on the criteria developed by Moynihan et al.37 This includes the four studies that used the Media Doctor Australia tool12,13,38,39 and the 3 studies that used the Health News Review tool,25,26,40 which was based on the Media Doctor Australia tool.25 Two studies used two separate tools.38,41 We did not identify any studies that used the Quality Index for health-related Media Reports9 or the checklist for improving drug information in the general press.10\n\nObjectives, sampling frames, selection criteria and technique, and reported subgroup analyses\n\nThe explicit or implicit objectives of studies included in the qualitative summary are presented in S3 Table, the sampling frames and selection criteria and techniques in S4 Table, and the variables used in the reported subgroup analyses in S5 Table (see extended data104). Detail and clarity varied. For example, some studies reported explicit inclusion and exclusion criteria for news reports, while others simply stated that they assessed news reports that included health or medical information.\n\nThirty-one studies (70%) reported at least one subgroup analysis, exploring differences in quality between subgroups of news reports. The most common overarching variables were ones related to time, medium, and outlet. How subgroups were categorised for the same overarching variable, as well as what categories were included, varied. For example, some studies included subgroup analyses based on specific intervention (e.g., comparing the quality news reports about one medicine with news reports about another medicine). Others included subgroup analyses based on the category of intervention (e.g., comparing news reports about “modern” medicines versus news reports about other categories of interventions). Furthermore, the specific interventions or categories of interventions varied.\n\nTwo reviewers assessed all 208 criteria applied to news reports in the 18 studies included in the meta-analyses. Not all criteria were measures of quality. Of the 208 criteria, 77 (37%) were excluded: 66 (32%) for being non-generic or descriptive, for measuring factual accuracy, or for not being clear or sensible (having inadequate face validity); and 11 (5%) for being global scores or composites. We provide examples in Table 3. Quality criteria were judged not to be sensible if they were in conflict with the IHC Key Concept and research evidence, for example a criterion assessing whether research presented in a news report is peer-reviewed.40,42 The same two reviewers mapped the remaining 131 of 208 quality criteria (63%) against the IHC Key Concepts. Based on the results of the mapping, we established 19 groups of quality criteria (criteria groups), as well as eight subgroups.\n\nIn S6 Table in the extended data,104 we present the number of quality criteria included in the meta-analyses per relevant IHC Key Concept. We were unable to identify or include quality criteria related to several IHC Key Concepts that are important for communicating information about the effects of health interventions in the news media or otherwise. For example, there were no criteria related to the concept “Average differences between treatments can be misleading” (2.3c). In contrast, a checklist for people communicating evidence-based information about the effects of healthcare interventions includes the item “Help your audience to avoid misinterpreting continuous outcome measures,” granted the checklist is not for journalists.43\n\nOf the 131 quality criteria included in the 19 groups, we excluded 22 (17%) from the meta-analyses for inadequate reporting of results, meaning it was impossible to impute the proportion of relevant news reports that satisfied the criterion based on the reported data. The distribution of the remaining 108 criteria amongst the 19 main groups and eight subgroups, is presented in Table 4, with the order of the groups based on the IHC Key Concept framework. In one case, we included a criterion in two groups: the third criterion in the study by Stassen,40 which we included in Criteria related to qualitative descriptions of effects and Criteria related to pros and cons. Several criteria from the same study were included in three of the 19 main groups (16%) and four of the eight subgroups (50%). Criteria related to “disease mongering”12 stem from a paper by Moynihan et al.44\n\nN = criteria included in the meta-analyses.\n\na Includes multiple criteria from the same study.\n\nThe primary meta-analyses are summarised in Figure 3, excluding subgroups. The samples of news reports in two of the studies included in the meta-analyses overlap.12,13 For groups that included a criterion from both of these studies, we performed primary analyses that included only the largest and most-recent study by Bonevski et al.,13 and an ad hoc sensitivity analysis that also included results from the study by Smith et al.12 The sensitivity analyses are summarised in Figure 4, again excluding subgroups. There were no important differences between the overall estimates and CIs for the primary analyses versus the sensitivity analyses. Forest plots for each criteria group, including individual estimates and CIs for each criterion, are presented in S7 File in the extended data.104\n\nFor most of the main groups (14 of 19) and a minority of the subgroups (two of eight), we did not consider it sensible to report overall estimates because—although the criteria in the group are clearly related—what they each measure was too different for it to be sensible to synthesise results. For example, in the group Criteria related to associations and randomization, the ninth criterion from the study by Bubela et al. measured whether the news report specifies that a trial was randomized or not, whereas the 12th criterion from the study by Haneef et al. measures whether there is a causal claim based on a non-randomized study. The two are clearly related, but at the same time, synthesising results would clearly not be sensible. Leaving out whether a study was randomized might be misleading by omission, but this can be compensated for by distinguishing causation and association and pointing out potential confounding. On the other hand, making a causal claim based on a non-randomized study is directly misleading. Further, we do not report overall estimates for the groups that include a single criterion.\n\nDue to heterogeneity, as well as inadequate reporting and small sample sizes in some studies, we did not conduct any of the six subgroup analyses considered in the protocol, exploring differences in quality between news reports:\n\n1. in different media (broadcast vs. other),\n\n2. from different decades,\n\n3. by commercial vs. non-commercial outlets,\n\n4. by health or science journalists vs. others,\n\n5. in low-income countries vs. middle or high-income countries, or\n\n6. in broadsheet vs tabloid newspapers.5\n\nRisk of bias\n\nThe individual risk of bias assessments for each of the 108 criteria included in the meta-analyses are presented in S9 File in the extended data.104 We considered there to be a high risk of bias in 13 cases (11%), including in the case of the one criterion included in two criteria groups (the third criterion in the study by Stassen40). The 13 criteria for which we considered there to be a high risk of bias are the 13 that required substantial judgement to be applied, according to our assessment. In 12 of those 13 cases, it was—in addition—unclear whether researchers were blinded to the publication and journalist behind the news report. In the remaining case, it was clear that they were not blinded. In four of the 13 cases, it was also unclear whether the criterion was applied independently by at least two researchers.\n\nQuality of the evidence\n\nWe only assessed the quality of the evidence for the five main groups and six subgroups where we report an overall estimate. Quality varied from low to high. In all but two of 10 cases, we downgraded quality for substantial inconsistency. In half of the cases, we downgraded for important imprecision. Finally, for the group criteria related to “disease mongering”, we downgraded for overall high risk of bias.\n\nWe summarise the main findings here. Details of these analyses are reported in S10 File in the extended data.104\n\nCriteria related to associations and randomization\n\nMany news reports included causal claims based on associations, and few said whether trials were randomized. We included three estimates, from three studies,19,32,45 related to the IHC Key Concepts that “An outcome may be associated with a treatment but not caused by it” (1.2d) and “Comparison groups should be as similar as possible” (2.1a). Based on estimates for single criteria with a low risk of bias, 51% of news reports make causal claims based on non-randomized studies (95% CI 40%-62%)32 and 9% specify whether a given trial was randomized (95% CI 7%-12%).19\n\nCriteria related to the need for comparisons\n\nSome news reports concluded that an intervention has an effect despite a lack of a comparator, and many did not include evidence to support the effectiveness of the intervention. We included three estimates, from three studies,12,32,38 related to the IHC key concept that “Identifying effects of treatments depends on making comparisons” (1.2f). Based on an estimate from a single study, three quarters (74%) of news reports avoid concluding there is a beneficial effect when there is a lack of a comparator (95% CI 63%-83%).32 Two studies found that only a third (34%)12 to half (55%)38 of news reports include evidence about the effects of the intervention (95% CIs 25%-43% and 38%-72%). The risk of bias for these two criteria was high.\n\nCriteria related to consistency between studies\n\nFew news reports considered and soundly assessed the consistency of evidence. We included a single criterion, from the study by Krauth and Apollonio,28 related to the IHC Key Concept that “The results of one study considered in isolation can be misleading” (1.2g) and “Consider how certain you can be about each advantage and disadvantage” (3.3d). The risk of bias was low for this criterion. The study found that 13% (95% CI 7%-26%) of news reports published in American newspapers and magazines about tobacco cessation therapy “consider” and make a “well-founded” assessment of consistency between studies.\n\nCriteria related to “disease mongering”\n\nSome news reports committed disease mongering. We included five estimates, from five studies,12,13,26,38,40 related to disease mongering, which is addressed by the IHC Key Concept that “Earlier detection of ‘disease’ is not necessarily better” (1.2k). All five criteria had a high risk of bias. Overall, 86% of news reports do not commit disease mongering (95% CI 78%-91%) (Figure 3). The certainty of the evidence for that estimate is low due to substantial inconsistency (I2 = 92%) and the high risk of bias.\n\nCriteria related to conflicts of interest\n\nMany news reports failed to address conflicts of interest. We included seven estimates, from seven studies,19,26,27,37,38,40,46 related to the IHC Key Concept that “Competing interests may result in misleading claims” (1.3b) All criteria had a low risk of bias. Overall, 22% (95% CI 7%-49%) in some way disclose potential conflicts of interest (Figure 3). The certainty of the evidence for that estimate is low, due to substantial inconsistency (I2 = 99%) and important imprecision.\n\nCriteria related to opinions\n\nSome news reports failed to adequately distinguish opinion and fact. We included a single estimate, from the study by Krauth and Apollonio,28 related to the IHC Key Concept that “Opinions alone are not a reliable basis for claims” (1.3d). The study evaluated American newspaper and magazine reports about tobacco cessation therapy. It found that three quarters (73%) of news reports adequately distinguish opinion and fact (95% CI 6%-83%). The criterion had a low risk of bias.\n\nCriteria related to study design and risk of bias in general\n\nMany news reports did not address the “quality of evidence”. We included five estimates, from five studies,13,26,28,38,40 related to the risk of bias (“type”, “credibility” or “quality” of the evidence), which is addressed by multiple low-level IHC Key Concepts under the high-level concepts “Comparisons of treatments should be fair” (2.1) and “Syntheses of studies need to be reliable” (2.2). Three of the criteria had a high risk of bias. The proportion of the news reports that meet the criterion used in each study varied from 8% (95% CI 3%-20%)28 to 77% (95% CI 73%-81%).40\n\nCriteria related to blinding\n\nFew news reports said whether trials were “double blinded”. We included a single criterion, from the study by Bubela et al.,19 related to the IHC Key Concepts that “The people being compared should be cared for similarly apart from the treatments being studied” (2.1c), “If possible, people should not know which of the treatments being compared they are receiving” (2.1d) and “Outcomes should be assessed in the same way in all the groups being compared” (2.1e). The study evaluated reports in newspapers in English-speaking countries about herbal remedy and conventional pharmaceutical trials. It found that 7% (95% CI 6%-9%) of news reports specify if a trial was “double-blinded”. The criterion had a low risk of bias. The term “double blind” is a problematic term since it can have several meanings.37 The study did not address this.\n\nCriteria related to loss to follow-up\n\nFew news reports said anything about follow-up of participants in trials. We included a single criterion, from the study by Bubela et al.,19 related to the IHC Key Concept that “It is important to assess outcomes in all (or nearly all) the people in a study” (2.1g). This was the same study that evaluated whether news reports specify whether a trial was “double blind”.18 The risk of bias was low for this criterion. The study found that 7% (95% CI 5%-9%) of news reports specify withdrawals or dropouts from a trial.\n\nCriteria related to selective reporting\n\nFew news reports selectively reported “statistically significant” results. We included two estimates, from two studies,32,45 related to IHC Key Concepts that “Failure to consider unpublished results of fair comparisons may result in estimates of effects that are misleading” (2.2b) and “Deeming results to be ‘statistically significant’ or ‘nonsignificant’ can be misleading” (2.3g). Overall, 93% (95% CI 87%-97%) of news reports avoid selectively reporting “statistically significant” results (Figure 3). The certainty of the evidence for that estimate is high.\n\nCriteria related to qualitative descriptions of effects\n\nMany news reports used misleading language to describe intervention effects and did not quantify effects. We included seven estimates, from five studies,26,27,32,37,40 related to the IHC Key Concept that “Verbal descriptions of the size of effects alone can be misleading” (2.3a) Overall, four studies that reported six criteria related to quantification of effects found that 53% (95% CI 36%-69%) of news reports quantify effects, as opposed to only describing them qualitatively. There was a high risk of bias for two of the criteria. The certainty of the evidence for this estimate is also low, due to substantial inconsistency (I2 = 99%) and important imprecision. Overall, two studies, found that 68% (95% CI 35%-90% of news reports do not use misleading language to describe intervention effects. One of the criteria had a risk of bias. The certainty of the evidence for that estimate is low, due to substantial inconsistency (I2 = 98%) and important imprecision.\n\nCriteria related to absolute effects\n\nMost news reports did not report absolute effects. We included five estimates, from five studies,12,13,37,38,47 related to the IHC Key Concept that “Relative effects of treatments alone can be misleading” (2.3b). All five criteria had a low risk of bias. Overall, 17% (95% CI 4%-49%) of news reports report absolute effects (Figure 3). The certainty of the evidence for that estimate is low, due to substantial inconsistency (I2 = 97%) and important imprecision.\n\nCriteria related to the play of chance\n\nFew news reports claimed that an intervention has a benefit despite a small sample size, and more than half specified the sample size of trials, but few clearly and soundly “assessed the precision”8 of reported estimates of effect. We included four estimates, from four studies,19,28,32,36 related to the IHC Key Concepts that “Small studies may be misleading” (2.3d) and “The use of p-values may be misleading; confidence intervals are more informative” (2.3f). Based on estimates from single studies 95% (95% CI 88%-98%) of news reports do not claim there was a benefit despite a small sample size,32 and 63% (95% CI 59%-67%) specify the sample size of trials.19 Two studies found that 6% (95% CI 1%-38%)36 and 8% (95% CI 3%-20%)28 of news reports “clearly and soundly” assess the precision of estimates or risk of random error.\n\nCriteria related to subgroup analyses\n\nMost news reports did not focus on inappropriate subgroups. We included a single estimate, from the study by Yavchitz et al.,45 related to the IHC Key Concept that “Results for a selected group of people within a study can be misleading” (2.3e). The study found that 94% (95% CI 85%-98%) of news reports avoid focusing on an “inappropriate subgroup”. The criterion had a low risk of bias.\n\nCriteria related to lack of evidence\n\nMost news reports avoided claiming that interventions have equivalent effects based on a difference that was not “statistically significant”. We included a single estimate, from the study by Yavchitz et al.,45 related to the IHC Key Concept that “Lack of evidence of a difference is not the same as evidence of ‘no difference’” (2.3h). The study found that 93% (95% CI 83%-97%) of news reports do not claim equivalence when there was a “statistically nonsignificant” difference.45 The criterion had a low risk of bias.\n\nCriteria related to options\n\nMost news reports did not include information about alternative interventions. We included seven estimates, from six studies,13,26,38,40,48 related to the IHC Key Concept “Be clear about what the problem or goal is and what the options are” (3.1a). Overall, 36% (95% CI 26%-47%) of news reports include information about alternative interventions (Figure 3). The certainty of the evidence for that estimate is moderate, due to substantial inconsistency (I2 = 96%).\n\nCriteria related to extrapolations\n\nSome news reports focused on surrogate outcomes or extrapolated from surrogate to important outcomes. Some news reports did not report indications for interventions and most did not report contraindications. Few news reports did not clearly report to whom the information applied. Some extrapolated from study participants to a larger or different population. Many news reports did not consider the availability of the intervention or the dosage of the intervention. Some interventions extrapolated from the study intervention to a different intervention.\n\nAltogether, we included 21 criteria, from 12 studies,12,13,19,26,28,32,35,38,40,45,48,49 related to the IHC Key Concepts that “Attention should focus on all important effects of treatments, and not surrogate outcomes” (3.2a), “Fair comparisons of treatments in animals or highly selected groups of people may not be relevant” (3.2b), “The treatments compared should be similar to those of interest” (3.2c) and “There should not be important differences between the circumstances in which the treatments were compared and those of interest” (3.2d). There was a low risk of bias for all the criteria.\n\nWe included two criteria, from two studies32,48 related to the IHC Key Concept that “Attention should focus on all important effects of treatments, and not surrogate outcomes” (3.2a). Overall, 82% (95% CI 76%-86%) of news reports do not solely focus on surrogate outcomes or extrapolate from surrogate to important outcomes. The certainty of the evidence for this estimate is high.\n\nWe included eight criteria, from six studies,28,32,35,38,48,49 related to IHC Key Concept that “Fair comparisons of treatments in animals or highly selected groups of people may not be relevant” (3.2b). Five of these criteria, from three of the studies,38,48,49 addressed reporting of indications and contraindications. Indications are reported in 70% (95% CI 64%-76%) to 97% (95% CI, 95%-98%) of news reports, whereas contraindications are only reported in 4% (95% CI 2%-8%) to 13% (95% CI 11%-16%) of news reports. Two criteria, from two studies,28,35 addressed whether it was clear to whom the information applies. These criteria are met in 94% (95% CI 81%-98%) to 100% (95% CI 98%-100%) of news reports. The other criterion, from the study by Haneef et al.,32 assessed whether results were extrapolated from study participants to a larger or different population. The study found that 85% (95% CI 72%-92%) of news reports do not extrapolate to a larger or different population.\n\nWe included eight criteria, from eight studies,13,19,26,32,38,40,49 related to the IHC Key Concept that “The treatments compared should be similar to those of interest” (3.2c). Five of these criteria addressed availability of the intervention. The availability of the intervention is reported in 41% (95% CI 37%-45%) to 79% (95% CI 66%-88%) of news reports. Two of the criteria addressed dosage. Those studies found that 14% (95% CI 11%-17%)19 to 50% (95% CI 46%-54%)49 of news reports specify or mention dosage. The other criterion addressed extrapolation from the study intervention to a different intervention. That study32 found that 85% (95% CI 72%-92%) of news reports do not extrapolate to a different intervention.\n\nCriteria related to animal studies\n\nSome news reports extrapolated a beneficial effect from an animal study to humans. We included a single estimate, from the study by Haneef et al.,32 related to the IHC Key Concept that “Fair comparisons of treatments in animals or highly selected groups of people may not be relevant” (3.2b). The study, which had a sample of 29 news reports, found that 79% (95% CI 61%-90%) of reports do not extrapolate beneficial effects in humans from animal studies.32 The criterion had a low risk of bias.\n\nCriteria related to pros and cons\n\nFew news reports failed to mention at least one benefit of an intervention. On the other hand, few mentioned or discussed the cost of the intervention, and most did not mention or adequately discuss or explain potential harms.\n\nAltogether, we included 32 estimates, from 17 studies,12,13,26–28,32,35–38,40,45–50 related to IHC Key Concepts that “Treatments can cause harms as well as benefits” (1.1a) and “Weigh the benefits and savings against the harms and costs of acting or not” (3.3a). One criterion was also included with criteria related to qualitative descriptions of effects, related to the IHC Key Concept that “Verbal descriptions of the size of effects alone can be misleading” (2.3a). That criterion was the only one that had a high risk of bias.\n\nWe included 14 criteria, from 13 studies,12,13,27,32,37,38,40,45–50 related to harms. Overall, 40% (95% CI 23%-61%) of news reports about the effects of health interventions mention or adequately discuss or explain potential harms of the intervention. The certainty of the evidence for that estimate is low, due to substantial inconsistency (I2 = 99%) and important imprecision.\n\nWe included 10 criteria, from nine studies,13,26,27,37,38,40,47–49 related to cost. Overall, 18% (95% CI 12%-28%) mention or discuss the cost of the intervention. The certainty of the evidence for that estimate is moderate, due to substantial inconsistency (I2 = 98%).\n\nWe included two criteria, from two studies,38,48 related to benefits. Overall, 97% (95% CI 56%-100%) of news reports mention at least one benefit. The certainty of the evidence for that estimate is moderate, due to imprecision.\n\n\nDiscussion\n\nOverall, we found that researchers have conducted many empirical studies of the quality of news reports about the effects of health interventions, assessing diverse samples of reports in terms of medium, country and categories of interventions, using many different tools and criteria. Some criteria did not make sense and some relevant aspects of quality were not covered. We were able to synthesis results for some criteria groups, showing that there were several prevalent and important problems with the quality of news reports about the effects of health interventions. Many of the reports gave an unbalanced and oversimplified picture of the potential consequences of the interventions, leaving out information about conflicts of interest, alternative interventions, potential harms, and costs, and failing to quantify effects in absolute terms or at all.\n\nSpecifically, we identified 44 primary studies for inclusion in the qualitative summary (see extended data - S1 Table104). Newspaper was the most common medium. Of the 39 studies that specified a medium, newspaper content was explicitly included in 35 (90%). The United States (US) was the most common country in which content was published. Of the 40 studies where at least one country was specified, 12 (30%) included content published in the US, while 25 (63%) included content published in English-speaking countries. “Modern” medicine was the most common category of health interventions. In the 32 samples where a category of intervention was specified, 24 (75%) included news reports about “modern” medicine. No study specified whether sampled reports were published by commercial or non-commercial outlets. Most studies used a tool that was original or modified.\n\nThe Index of Scientific Quality was the most common tool (set of criteria). It was used in seven studies (16%).28,30,31,33–36 However, a larger number of studies used tools directly or indirectly based on the criteria developed by Moynihan et al.,37 including the four studies that used the Media Doctor Australia tool12,13,38,39 and the three studies that used the Health News Review tool.25,26,40\n\nOf the 44 studies included in the qualitative summary, we included 18 (41%) in the meta-analyses. We mapped all 208 quality criteria used in those studies against the IHC Key Concepts. Some of the criteria were in conflict with IHC Key Concepts, such as those that asked whether research discussed in a news report was peer-reviewed. We were unable to identify or include quality criteria related to several highly relevant IHC Key Concepts, for example “Average differences between treatments can be misleading” (2.3c).\n\nIn the end, we included 108 of the 208 criteria (52%) in the meta-analyses, organised in 19 groups, as well as eight subgroups. We calculated overall estimates and rated the certainty of the evidence for five of the 19 main criteria groups (26%) and 6 of the 8 subgroups (75%). In those cases, we found:\n\n• 86% of news reports commit “disease mongering” (95% CI 78%-91%) (low certainty),\n\n• 22% adequately address conflicts of interest (95% CI 7%-49%) (low certainty),\n\n• 93% do not selectively report “statistically significant” results (95% CI 87%-97%) (high certainty),\n\n• 53% quantify effects (95% CI 36%-69%) (low certainty),\n\n• 68% do not use misleading language (95% CI 35%-90%) (low certainty),\n\n• 17% report absolute effects (95% CI 4%-49%) (low certainty),\n\n• 36% include adequate information about alternative interventions (95% CI 26%-47%) (moderate certainty),\n\n• 82% do not focus on surrogate outcomes or extrapolate from surrogate to important outcomes (95% CI 76%-86%) (high certainty),\n\n• 40% mention, discuss or explain potential harms of the intervention (95% CI 23%-61%) (low certainty),\n\n• 18% mention or discuss the cost of the intervention (95% CI 12%-28%) (moderate certainty), and\n\n• 97% mention at least one benefit (95% CI 56%-100%) (moderate certainty).\n\nMedia doctor Australia and health news review\n\nSome of the studies included in our review report partial results from initiatives aimed at helping journalists and the public assess health information in the news and improving health journalism. This includes studies related to the Media Doctor Australia initiative12,13,39 and its American counterpart Health News Review (or HealthNewsReview.org).25,26 These initiatives involved an editorial team rating news reports about the effects of health interventions on a running basis and posting individual ratings with lay explanations on a dedicated website. Both have been shut down. We were unable to include the most complete published results from either initiative in our meta-analyses, but those results were consistent with the earlier results that we were able to include, as well as our overall findings.\n\nIn terms of Media Doctor Australia, we were able to include the studies by Smith et al.12 and Bonevski et al.13 in the meta-analyses. The former includes results from the first 104 ratings, published between Feb. 1 and Sept. 1, 2004, while the latter includes results from the first 222 ratings of news reports about “complementary” and “alternative” medicine, published between Jan. 1, 2004, and Sept. 1, 2007. Meanwhile, Wilson et al. include results from 1230 ratings, posted between March 2004 and June 2008, in a paper titled “Media reporting of Health Interventions: Signs of Improvement, but Major Problems Persist”.39 We included the study by Wilson et al. in our qualitative summary, but not in the meta-analyses, since the authors only reported mean scores and not the sample sizes for each quality criterion. We knew from the two other Media Doctor Australia studies that sample sizes varied between criteria because a criterion was not always considered relevant to a given news report and, in those cases, not applied. In other words, we could not extract the exact proportions of news reports satisfying each criterion from the study by Wilson et al. That said, as the title of paper suggests, there were no notable differences between the new Media Doctor Australia results and the previous results reported by Smith et al. (see Table 2 in the study by Wilson and colleagues39).\n\nIn terms of Health News Review, we were able to include the studies by Schwitzer25 and Walsh-Childers et al.26 in the qualitative summary. The former included results from 500 Health News Review ratings, published between April 2006 and February 2008, while the latter included results from 1889 ratings published from July 2005 through March 2013. Hence, we only included the study by Walsh-Childers et al. in the meta-analyses. The Health News Review team continued to publish ratings until the end of 2019, finishing at 2610.51 In a blog post published Dec. 20, 2018, the founder and publisher, Schwitzer, shared a final “report card” with some of the results from all 2610 ratings.51 Like with the Media Doctor Australia studies, there were no notable differences between the most recent results, published in Schwitzer's blog post, and those previously reported and included in this review, from the study by Walsh-Childers et al.\n\nUnderlying problems\n\nProblems with the quality of information in health news reports could be explained by underlying problems occurring at different stages of the information pipeline, from when scientists plan, conduct and report studies, to when academic institutions and journals promote studies, to when news outlets cover studies. Not to mention deliberate misinformation by commercial industries and organisations.52\n\nIoannidis has estimated that most published research findings are false due to small studies and small effect sizes, testing many relationships without preselecting those tests, flexibility in study designs, definitions, outcomes and analyses, financial and other conflicts of interest, and competition.53 Similarly, Chalmers and Glasziou have estimated that 85% of research is wasted due to wrong research questions, unnecessary or poorly designed studies, failure to publish relevant research promptly or at all, and biased or unusable reports of research.54 Meanwhile, Chi and colleagues have found problems with the quality of information in biomedical literature that correspond with problems found in health news reporting, for example inappropriate use of causal language.55 A vast amount of poorly planned, conducted and reported research provides a terrible starting point for health and science journalism. Parenthetically, these studies provide additional evidence that peer review is a poor indicator of quality.\n\nSeveral studies have identified problems with press releases about health research. Wang et al. found randomised trials were less likely to be the subject of a medical journal press release compared to observational studies.56 Woloshin et al. assessed press releases from academic medical centres and found many were directly misleading or misleading by omission.57 Yavchitz et al. found press releases for two-arm, parallel-group randomised trials often included some form of “spin”, for example conflating lack of evidence of a difference with no difference.45 Sumner et al. have found exaggerations in press releases from both academic institutions58 and scientific journals,59 such as causal claims based on observational studies, were associated with exaggerations in related news reports. This finding was supported by a replication study conducted by Bratton et al.60\n\nAmend and Secko conducted a qualitative synthesis of studies exploring journalists’ experience of covering health and science.61 They included 21 studies involving 788 journalists in total. Many of the themes cutting across studies were barriers to high-quality health and science journalism, including deadline pressure and limited time for researching and preparing stories; limited time or space for telling stories; limited budgets and staff, including a lack of specialised health and science journalists; competition and commercialization; pressure from advertisers and interest groups; and lack of education or training.\n\nOther mass media sources of health information\n\nOther studies have focused on other mass media sources of lay information about the effects of health interventions. This includes both other forms of journalism, besides news reports, and other mass media, besides news media. Some of those studies were included in this review. Overall, it appears quality varies depending on the source, but that there are problems across the board, granted we have not systematically reviewed or critically appraised studies focusing on other sources.\n\nNewspaper features and advice columns – Heaner assessed news features and news reports about nutrition and physical activity.29 She found features were generally of higher quality, which is unsurprising given that they were on average twice as long and probably had longer deadlines. Molnar et al. assessed medical advice columns for elderly readers and found that recommendations were often inappropriate or potentially dangerous.62\n\nEntertainment – Wilson et al. assessed the quality of advice popular magazine articles and found especially those with “health” in the title of the publication presented poor-quality, unreliable advice.63 Korownyk et al. and Mishori et al. assessed recommendations made on medical talk shows.64,65 Both studies found the recommendations were often unreliable or misleading.\n\nSocial media – Moorhead et al. conducted a systematic review of the uses, benefits and limitations of social media for health communication and found that concern about the quality of information was common across studies. Li et al. found that over a quarter (19) of the 69 most-viewed YouTube videos about Covid-19 contained misleading information, including “inappropriate recommendations”, consistent with previous studies of YouTube videos about other pandemic diseases.66 Haber et al. found many of the health science articles most often shared on Twitter and Facebook in 2015, as well as media coverage of those articles, often used causal language that was too strong for the strength of the evidence.24\n\nAdvertising – There is a long tradition of misleading advertisements for health interventions. Dushman has provided 10 examples from the early 20th century, including an ad for Dr. William O. Coffee's “world-famous”, “marvelous” and “remarkable” treatment for deafness.67 Frosch et al. and Faerber and Kreling have found problems with modern advertisements for both prescription and over-the-counter medicines.68,69 In sales materials for herbal dietary supplements, the United States Government Accountability Office has found “improper” and illegal claims that the supplements can cure cancer, among other conditions.70\n\nOther consumer and patient sources – In a systematic review of studies assessing the quality of online health information for consumers, Eysenbach et al. found 55 of 79 studies (70%) had concluded that quality was a problem.71 In more recently published studies, others have found problems with the information on anti-vaccination websites,72 cancer and oncological industry websites,73 websites for clinics offering weight loss surgery,74 and websites for fertility centres.75 In their study of media coverage of practice-changing clinical trials in oncology, Andrew et al. found the cancer and industry websites provided higher-quality information than newspaper and cable news reports.73\n\nStudies focusing on patient sources of information have found problems with patient health portals,76 patient materials,77 and brochures from a health fair.31 Glenton and Oxman (AO) surveyed and interviewed leaders of organisations representing health care users.78 They concluded that the organisations did not appear to promote evidence-based health care and that when they did promote scientific information, they appeared to do so uncritically, relying on limited sources and traditional authorities.\n\nLast but not least, in a review published in 2019, Oxman and Paulsen assessed free, online sources of “trustworthy” information about the effects of health interventions.79 They found that patients and the public could access trustworthy information using two websites: Cochrane Evidence (www.cochrane.org/evidence)80 and Informed Health (www.informedhealth.org).81 However, they concluded that both websites could be improved by consistently reporting information about the size of both benefits and harms, and the certainty of the evidence. They noted that many websites excluded from their review claim to provide evidence-based or reliable information about the effects of health interventions, but that it was difficult to assess the reliability of the information on those websites since the information was not explicitly based on systematic reviews.\n\nOther problems with health news\n\nOther studies have found other problems with health news, besides the quality of information. Wang et al. found that observational studies and small randomised trials reporting surrogate outcomes generated as much news coverage as large, randomised trials reporting important outcomes.56 Dumas-Mallet et al. found that newspaper reports about biomedical research were typically about findings from initial studies.82 Such findings were often contradicted by a meta-analysis, but this was rarely reported by news outlets.\n\nStrengths\n\nAs far as we are aware, this is the first systematic review of the quality of any type of health news. It provides a larger, more detailed picture of the quality of health news than any primary study that we identified. It also provides a starting point for similar reviews and alternative analyses, and it highlights important issues for interpreting and planning new studies. We conducted an expansive search and identified several studies outside the peer-reviewed literature (see extended data - S1 Table104). We assessed records and full texts in any language for inclusion in the review, and included studies in five different languages in the qualitative summary (English, German, Norwegian, Slovenian and Spanish) (see extended data - S1 Table104). We synthesised results from up to 13 different studies, using different tools and criteria, and pooled results for up to 4116 unique news reports (see extended data - S8 and S10 Files104). The review provides an accessible summary of what is known about the quality of news reports about the effects of health interventions, in print, broadcast and online news media, for those aspects of quality that have been measured and reported. It also identifies gaps in what is known and directions for future research.\n\nLimitations\n\nThe findings of this review are limited to information about the effects of health interventions and “traditional” news media. In the section “Other mass media source of health information”, we reference and discuss findings from research focusing on other news media and other non-news media, but we have not systematically reviewed or critically appraised such studies. Furthermore, we did not explore applicability of the synthesised results to different types of traditional news media due to limited sample sizes, as well as inadequate reporting in the included studies.\n\nExamples of other types of health information include the accuracy of diagnostic tests and information about the health effects of exposures. News reports about the health effects of exposures (e.g. fatty foods) can be difficult to distinguish from news reports about the effects of health interventions (e.g. a low-fat diet). Some primary studies included in this review conflated the two, such as the study by Hackman and Moe, which assessed news about nutrition-related research.83 We included this study in the qualitative summary, but not the meta-analyses. We did this because key concepts for assessing or communicating information about the effects of interventions may not apply to the same degree, or at all, to information about associations between exposures and outcomes. For example, Woodruff and Sutton point out that randomised trials are “virtually precluded” from evidence about hazardous environmental exposures because of ethical considerations”.84 It is possible, if not likely, that there are important differences in quality between news reports about the effects of exposures versus interventions.\n\nWe excluded studies or criteria that were only descriptive, even though criteria that were descriptive sometimes provided the same data as criteria explicitly intended to assess quality. For example, Mercurio and Eliott describe Australian news about “alternative” medicine for cancer, but do not explicitly assess the quality of those news reports,85 so we excluded the study. However, one of their coding variables was “Mention of risks, benefits and costs”. The data for this variable would have fit with data in our meta-analyses, in the group Criteria related to pros and cons. We consider it unlikely that inclusion of studies or criteria that were only descriptive would have substantially altered the findings of this review. Similarly, we only included studies in the meta-analyses if they were in English or Norwegian (one of the languages spoken fluently by the primary investigator, MO), but consider it unlikely that the studies reported in other languages would have substantially altered the findings of this review either, given that the underlying problems discussed earlier on are unrelated to language.\n\nWe did not code raw data ourselves or contact authors for help where data were inadequately reported for our purposes. Finally, we did not consider which criteria were more informative, only whether they were eligible and sensible, nor did we consider statistical measurement properties.86\n\nImplications for different stakeholders are summarised in Table 5 and explained in more detail in the subsequent sections.\n\nImplications for journalists, editors, and news outlets\n\nThere is clearly room for improving the quality of health and science journalism. Based on our meta-analyses, journalists and editors should pay special attention to the following IHC Key Concepts (see extended data - S6 File104) when communicating information about the effects of health interventions:\n\n• “Treatments can cause harms as well as benefits” (1.1a),\n\n• “Competing interests may result in misleading claims” (1.3b),\n\n• “Verbal descriptions of the size of effects alone can be misleading” (2.3a),\n\n• “Relative effects of treatments alone can be misleading” (2.3b),\n\n• “Be clear about what the problem or goal is and what the options are” (3.1a), and\n\n• “Weigh the benefits and savings against the harms and costs of acting or not” (3.3a).\n\nBased on individual estimates from primary studies included in this review (see extended data - S8 and S10 Files104), there is likely also room for improvement related to other highly relevant IHC Key Concepts, such as “The treatments compared should be similar to those of interest” (3.2c). Moreover, there is likely room for improvement related to highly relevant IHC Key Concepts that were not captured by any quality criteria, such as “Average differences between treatments can be misleading” (2.3c) (see extended data - S10 File104).\n\nJournalists and editors should take a critical approach to reports of research, especially single studies and observational studies, and to press releases (see “Underlying problems”). When reporting on the effects of interventions, they can search the Cochrane Evidence (www.cochrane.org/evidence)80 and Informed Health (www.informedhealth.org)81 websites for summaries of relevant systematic reviews. If possible, news outlets should provide or support special training for health and science reporters, as well as provide adequate time for researching and preparing stories, and adequate time or space to tell stories.\n\nJournalists and editors who are interested in learning more about IHC Key Concepts can do so for free on the “That's a claim!” website (www.thatsaclaim.org),87 where all of the concepts are explained in plain language, and there are references to relevant learning resources for each individual concept. They can also find free learning resources for respective concepts via the Teachers of Evidence-Based Health Care portal (www.teachingebhc.org).88\n\nImplications for researchers, scientific journals, academic institutions, and research funders\n\nResearchers, journals, and academic institutions should pay special attention to the same IHC Key Concepts as journalists and editors, as well as other highly relevant concepts (see previous section), when reporting the results of studies assessing the effects of interventions in research papers and press releases, as well other contexts, such as conferences. More fundamentally, they should work to reduce false findings and research waste. This also goes for organisations that fund research.\n\nThe results of this review have a series of implications for investigators planning new studies of the quality of information about the effects of health interventions in mass media (as well as peer reviewers assessing new studies), from objectives, to methods, to reporting. First, they should think carefully about their goal. New studies can be designed to have practical value, for example informing interventions to improve a particular population's ability to think critically about health information by identifying the IHC Key Concepts that are most relevant for that population—e.g. a study of health claims in advertisements targeted at young people. In addition, they should consider research gaps that we have revealed. Again, there does not appear to be studies assessing the quality of health news reports in terms of several highly relevant IHC Key Concepts (see extended data - S6 Table104). Neither does there appear to be studies that compare the quality of reports published by commercial outlets versus public or independent outlets. Others have pointed out that research is needed about how best to communicate information about the effects of health interventions,43 including how to communicate uncertainty.89\n\nSecond, they should think carefully about what tools and quality criteria to use, whether using a previously developed tool, adapting one, developing a new tool, or some combination. They should avoid criteria that are overly general (e.g. global scores and composites) or unclear (e.g. criteria assessing whether “double blinding” is mentioned), and criteria that do not make sense (e.g. criteria assessing whether research was peer-reviewed). They should be deliberate about the trade-offs between how straightforward it is to apply a criterion and report the results versus how informative it is. For example, it is relatively easy to see whether a news report mentions the design of a study. It is more difficult, but also more informative, to assess whether the report adequately discusses strengths and limitations of said design.\n\nThird, regarding samples of news reports, they should consider applicability and potential explanatory factors, for example those we highlighted in the protocol for this review (see extended data - S1 File104) as possible variables for subgroup analyses: medium; time period; financial model (commercial vs. non-commercial); specialisation (specialised health or science journalist vs. other); country income level; and newspaper type (broadsheet vs. tabloid). Fourth, they should consider risk of bias. If possible, two researchers should assess each news report or other unit of mass media content, and the researchers should be blinded to factors that could introduce bias, such as the name of the journalist or publication. This is especially important for criteria that require substantial judgement.\n\nFifth, when reporting new studies, researchers should be explicit, clear and comprehensive. They should include the study objective, the sampling frame, and the selection criteria and technique, with concern for applicability and comparing results across studies, including results for subgroups. They should include the time period in which the content was published, as well as when it was assessed; the country in which it was published or, if online content, the country that the outlet primarily targets or where it is based; the categories of interventions that the content focuses on; and the financial models of the outlets. If using a previously developed tool, they should provide reference to where the development is described, note whether they made any adjustments and, if so, explain them. If using a new tool, they should clearly describe how it was developed and what informed it, possibly in a separate paper. If the researchers received any training in applying the criteria, they should describe what this entailed. Finally, they should report results for each quality criterion separately, not just mean scores, and report absolute numbers (the total number of news reports or other units to which the criterion was applied and the number of units that satisfied the criterion), including for subgroups.\n\nImplications for consumers and patients\n\nThe implications for consumers and patients are similar to those for journalists and editors. They should pay special attention to the same IHC Key Concepts, as well as other highly relevant concepts (see “Implications for journalists”); they should approach health information in the mass media critically; they can refer to the Informed Choices and Cochrane Evidence websites for summaries of relevant systematic reviews; and they can take advantage of the “That's a claim!” website and Teachers of Evidence-Based Health Care portal to learn more about IHC Key Concepts.\n\nImplications for citizens and policymakers\n\nThe results of this review, as well as the results of related studies referenced in the section “Other mass media sources of health information”, show that the global “infodemic” was compounded by Covid-19, but started long before the World Health Organization coined the term in their situation report on the virus published Feb. 13, 2020.3 The infodemic is so dangerous because we know so many people are unable to critically assess claims or evidence about the effects of health interventions.90,91 When people act on those claims or beliefs that are unreliable or fail to act on reliable advice, they may suffer unnecessarily or waste resources. It follows that infodemic is probably an important factor in the enormous, worldwide overuse of ineffective or harmful interventions92 and underuse of effective interventions.93 Even one misleading news report might contribute to wasteful, harmful, and even deadly choices. A dramatic example of misleading health news is coverage of the baseless suggestion that the measles, mumps and rubella vaccine causes autism.94 Incidentally, news reports often focus on the benefits of interventions while ignoring or downplaying the harms, but this example illustrates that unbalanced reporting can also go in the opposite direction.\n\nIf the only goal of news about the effects of health interventions was to facilitate well-informed health choices, the content and format might look more like Cochrane plain language summaries, which have been systematically developed towards exactly said end.95,96 The reality is that news outlets existentially depend on getting people's attention, not informing them. Commercial media rely on advertising, not least advertisements for health interventions, which provides incentive to sensationalise. In turn, this can create pressure on competing public and independent outlets to do the same. Along the same lines, the raison d'être of press releases is inciting media coverage, while scientists are forced to “publish or perish”. Setting aside systemic barriers to reliable health news (see the section “Underlying problems”), it may be difficult if not impossible for journalists to produce news reports that satisfy all sensible, applicable criteria identified in this review in a report that people would understand and want to read, not to mention other potential criteria related to other highly relevant IHC Key Concepts. Even if a report includes and appropriately presents all relevant information about an intervention, it is possible that many people will not read more than the headline, missing important details, such as potential side effects.\n\nIt is possible for news outlets, including commercial ones, to improve their health and science coverage without shrinking their audiences and perhaps even increasing their numbers of readers, listeners, or viewers in some cases. But putting an end to unreliable health news is a Sisyphean task, not to mention health misinformation from other sources. In 2009, during a debate titled “Does the media support or sabotage health?”, Goldacre suggested: “The future of people getting information about health does not lie in journalism but people having direct access to information or the world of blogs where people can link direct to primary sources. There is no role for the health journalists of the future other than as entertainers\".97 However, it seems unlikely that giving people more health information—even if it is reliable—will solve the problem, when they are already overwhelmed and lack the skills needed to “separate the wheat from the chaff”.\n\nAs the saying goes, give someone a fish and you will feed them for a day; teach them to fish and you feed them for a lifetime. In other words, you can provide people with reliable health information or advice, and maybe they will believe you. If you help them understand why it is reliable, or why a claim is unreliable, you are preparing them to assess information and make informed choices for themselves in the future, inoculating them against unreliable claims and uninformed choices. Hence, we should be helping people gain basic critical appraisal and decision-making skills, in addition to providing access to reliable information. Moreover, by improving people's ability to assess evidence or recognise a lack of reliable evidence, the public may start to demand reliable studies, reducing false findings and research waste.\n\nFocusing on the education system allows for reaching large groups of people, gathered specifically to learn. It is important to start young, when people have time to reinforce and build upon what they have learned, and they have less to unlearn. The Informed Health Choices network, which includes authors of this review, has developed respective interventions to help primary school children and university students master select IHC Key Concepts, and has started to develop an intervention for secondary school students as of writing (www.informedhealthchoices.org/secondary-school-resources).98\n\nEducational interventions, like health intervention should be rigorously evaluated to find their effects, both positive and negative.99–101 The IHC team evaluated the primary school intervention in a cluster-randomised trial in Uganda, involving more than 10,000 children. The results showed that even children in a low-income setting, with large class sizes (on average 84 children in both intervention and control schools at the beginning of the trial), can learn to apply IHC Key Concepts, and the children retained what they learned for at least a year.102 Importantly, the intervention was developed using a human-centred design approach.103 A process evaluation linked to the trial showed that children, teachers, headteachers and parents were positive to the project and supported expanding it to other populations.104\n\nThe Norwegian Bak overskriftene (“Behind the headlines”) intervention targeted at university students takes advantage of health claims in the mass media.105 In the first instance, it has been implemented in a mandatory, introductory course on evidence-based health care, for students in the first two years of all health sciences bachelor programmes at Oslo Metropolitan University (about 1600 students per year). The intervention exploits that media content by design is simple, familiar, relatable, and entertaining, making it a favourable starting point for learning about critical appraisal compared to scientific literature, which typically includes jargon and excludes narrative. Journalism school students and staff have helped develop the intervention, which could potentially be used to train both journalism students and professionals, thereby increasing the quality of health and science news, assuming the intervention is properly evaluated and found effective.\n\nFinally, mastering IHC Key Concepts might help people assess information and make informed choices about non-health interventions as well. In collaboration with colleagues from other disciplines, the IHC team has established that most of the concepts apply to at least 13 other fields: agriculture, economics, education, environmental management, international development, informal learning, management, nutrition, planetary health, policing, speech and language therapy, social welfare, and veterinary medicine.100\n\n\nData availability\n\nAll data underlying the results are available as part of the article and its supporting information files and tables.\n\nZenodo: Quality of news reports about the effects of health interventions - Supporting Information. http://doi.org/10.5281/zenodo.4781182.104\n\nThis project contains the following extended data:\n\n- S1 File. Protocol. “Quality of news media reports about the effects and costs of health interventions: Systematic review protocol”.\n\n- S2 File. Protocol Appendix 1. “Appendix 1: Reference list of potentially eligible studies”.\n\n- S3 File. Protocol Appendix 2. “Appendix 2: Description of search for an existing review”.\n\n- S4 File. Dissertation. “Criteria used to measure the quality of news media reports about the effects of health interventions: Systematic review”.\n\n- S5 File. Detailed search strategy.\n\n- S6 File. Informed Health Choices Key Concepts. 2019 version.\n\n- S7 File. Detailed information about deviations from the protocol.\n\n- S8 File. Forest plots for individual criteria groups.\n\n- S9 File. Individual risk of bias assessments.\n\n- S10 File. Individual criteria included in meta-analyses. Sample characteristics, verbatim and reworded criteria, related IHC Key Concepts, overall risk of bias, estimates and confidence intervals, and sample sizes.\n\n- S1 Table. Included studies. References for all studies included in qualitative summary and reason for exclusion from meta-analyses or number of criteria included in meta-analyses.\n\n- S2 Table. Sample characteristics and tools by study. Sample size, medium/media, country/countries, time period(s), intervention category/categories, and tool(s).\n\n- S3 Table. Objectives of included studies.\n\n- S4 Table. Sampling frames and methods.\n\n- S5 Table. Reported subgroup analyses.\n\n- S6 Table. Number of quality criteria included in the meta-analysis per relevant IHC Key Concept.\n\nZenodo: PRISMA checklist for ‘Quality of information in news media reports about the effects of health interventions: systematic review and meta-analyses’. http://doi.org/10.5281/zenodo.4781182.104\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\nMO drafted the protocol and this paper. AO provided feedback on the draft protocol. All co-authors provided feedback on drafts of this paper. MO, LL and AQ piloted the eligibility criteria for primary studies. MO conducted all database searches, including citation searches. MO, LL, GG, DA, AQ and KB checked reference lists. All authors apart from CR contributed to study selection. All authors apart from AO and CR contributed to data extraction. MO and AO assessed, mapped and grouped the quality criteria. CR conducted the meta-analyses. MO and LL assessed risk of bias. MO and AO rated the certainty of the evidence.", "appendix": "Acknowledgements\n\nMike Clarke provided early guidance in a course on systematic reviews. Carl Heneghan and Kamal Mahtani were MO's MSc supervisors and provided feedback on the protocol and the first part of the review (MO's dissertation). Marit Johansen provided feedback on the search strategy. Anette Blüme, Tina Poklepović Peričić, Rob J.P.M. Scholten and Michel Wensing helped assess full texts in German, Slovenian and Dutch for inclusion in the qualitative summary. We are grateful to Mark Oette for providing a copy of his study, included in the qualitative summary. 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PubMed Abstract \| Publisher Full Text\n\nMokkink LB, Prinsen CA, Patrick DL, et al.: COSMIN manual for systematic reviews of PROMs, user manual.2018: 1–78. Reference Source\n\nCentre for Informed Health Choices, InfoDesignLab, Epistemonikos Foundation: That's a claim![cited 18 Feb 2021]. Reference Source\n\nInternational Society for Evidence-Based Health Care: Teachers of Evidence-Based Health Care.[cited 18 Feb 2021]. Reference Source\n\nBüchter RB, Betsch C, Ehrlich M, et al.: Communicating Uncertainty in Written Consumer Health Information to the Public: Parallel-Group, Web-Based Randomized Controlled Trial. J Med Internet Res. 2020; 22: e15899. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoutron I, Haneef R, Yavchitz A, et al.: Three randomized controlled trials evaluating the impact of “spin” in health news stories reporting studies of pharmacologic treatments on patients’/caregivers’ interpretation of treatment benefit. BMC Med. 2019; 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDahlgren A, Furuseth-Olsen K, Rose CJ, et al.: The Norwegian public's ability to assess treatment claims: results of a cross-sectional study of critical health literacy. F1000Res. 2020; 9: 179. Publisher Full Text\n\nBrownlee S, Chalkidou K, Doust J, et al.: Evidence for overuse of medical services around the world. Lancet. 2017; 390: 156–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlasziou P, Straus S, Brownlee S, et al.: Evidence for underuse of effective medical services around the world. Lancet. 2017; 390: 169–177. PubMed Abstract \| Publisher Full Text\n\nGoldacre B: Bad science. London: Fourth Estate; 2008.\n\nSantesso N, Rader T, Nilsen ES, et al.: A summary to communicate evidence from systematic reviews to the public improved understanding and accessibility of information: a randomized controlled trial. J Clin Epidemiol. 2015; 68: 182–190. PubMed Abstract \| Publisher Full Text\n\nCochrane Community: Cochrane begins project to improve Plain Language Summaries.18 May 2020 [cited 18 Feb 2021]. Reference Source\n\nCoombes R: Health journalism: two clicks away from Britney? BMJ. 2009; 338. Publisher Full Text\n\nRosenbaum S, Oxman M, Oxman A, et al.: Human-centred design development of Informed Health Choices (IHC) learning resources for secondary school students: Protocol.2019. Reference Source\n\nSharples JM, Oxman AD, Mahtani KR, et al.: Critical thinking in healthcare and education. BMJ. 2017; 357: j2234. PubMed Abstract \| Publisher Full Text\n\nAronson JK, Barends E, Boruch R, et al.: Key concepts for making informed choices. Nature. 2019; 572: 303–306. PubMed Abstract \| Publisher Full Text\n\nZhao Y: What works may hurt: Side effects in education. J Educ Chang. 2017; 18: 1–19. Publisher Full Text\n\nNsangi A, Semakula D, Oxman AD, et al.: Effects of the Informed Health Choices primary school intervention on the ability of children in Uganda to assess the reliability of claims about treatment effects, 1-year follow-up: A cluster-randomised trial. Trials. 2020; 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNsangi A, Semakula D, Rosenbaum SE, 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. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNsangi A, Semakula D, Glenton C, et al.: Informed health choices intervention to teach primary school children in low-income countries to assess claims about treatment effects: Process evaluation. BMJ Open. 2019; 9: e030787. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOxman M, Habib L, Jamtvedt G, et al.: Using claims in the media to teach essential concepts for evidence-based healthcare. BMJ Evidence-Based Med. 2020; 0: 1–3. PubMed Abstract \| Publisher Full Text\n\nRoberts S: Dr. Lisa Schwartz, Critic of Medical Excess, Is Dead at 55. In: The New York Times [Internet]. 6 Dec 2018 [cited 16 Feb 2021]. Reference Source" }	[ { "id": "89219", "date": "29 Jul 2021", "name": "Raymond Moynihan", "expertise": [ "Reviewer Expertise Medicine and Media", "Conflicts of Interest", "Overdiagnosis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments.\nThis review is extremely valuable – the first summarizing evidence about the quality of news reports about health interventions. I have a few short general comments and some specific comments.\nIn relation to the use of the word “qualitative” - I wonder if the authors might consider using a different word – or adding more explanation – perhaps saying “qualitative summary of results” – as it sounds to the casual reader like when you refer to “qualitative” you may have done a SR of “qualitative” studies of news coverage (i.e. focus groups with journalists etc.) – whereas I think you have a different meaning in mind. (e.g. P8 “qualitative summary”, which is also mentioned in Abstract).\nRe: IHC. Given the importance of IHC concepts for this SR, and the role of the authors in IHC world, and the advocacy of IHC in the Discussion, I think the IHC concepts etc. needs to be mentioned in the Introduction and expanded on in the Methods (as to how and why exactly the IHC concepts interacted with your analysis in the SR) - and the authors link to IHC can be mentioned in Methods somewhere. This is important and will help explain how the Discussion section becomes a little like advocacy for IHC materials.\nThe paper is long and complex – but with much valuable information. If any more material can be moved into Supp files, that will be helpful.\nI have no biostats training so I have not assessed the stats.\nSpecific comments.\nSearch strategy: It seems almost 2 years since it was updated. Authors might consider updating before indexing.\n\nP5: “We excluded inappropriate criteria…’\n\nPerhaps “ineligible” might be better?\n\nP7: Re: your sentences “Furthermore, we reframed negative quality criteria as positive, and paraphrased all quality criteria, for the data to be consistently framed and worded, and for the criteria to be succinctly labelled. For example, Bubela et al. report the number of news reports that “did not” specify whether a study was randomized, which was reframed as the number of news reports that did specify whether a study was randomized.”\n\nWhile this makes sense, I presume that sometimes that re-framing was not straight forward – and I wonder if there is anything more to say on any complexities associated with this approach. Also, if you can give a little more detail in the Bubela example, with numbers, that could help the reader.\n\nP7: “Risk of Bias”. Can the authors add a few words in text explaining why the \"risk of bias\" was solely about the risk of detection bias? A reader of an SR might normally expect a few different domains, so it would be good to just explain why this single domain was most relevant.\n\nP10: Table 2. I think Table 2 needs re-thinking – as it took me some time to work out what was going on. I was treating each row as a discreet coherent entity, but in fact you have multiple parts to each row. I am sure you can make it clearer somehow.\n\nP11: “In contrast, a checklist for people communicating evidence-based information about the effects of healthcare interventions includes the item “Help your audience to avoid misinterpreting continuous outcome measures,” granted the checklist is not for journalists.” I don’t fully understand meaning of this sentence - perhaps be a little clearer?\n\nP12: This is a structural suggestion. By the sub-heading “Synthesis of Results” I am really wanting to hear some of the key findings – in terms of content (i.e. a strong compelling prose version of the data in Figure 3) – but I feel I am still getting a lot of complex fine detail about the characteristics of studies and elements of them. I understand there is a lot to report – but some clear statements of the key findings – even summarised and abstracted a little – would be helpful around here to orient the reader.\nAlso – a related point - I find the three paragraphs on P12 (and into P13) complex and hard to follow. I wonder whether this level of detail is necessary in the main text – and whether it might be moved to a supplementary – but that is for authors to decide. At the very least – if there is any way to make it all a little clearer around this point – that would be great for the reader. An example of the mention of the “ninth criteria” from Bubela and 12th from Haneef. I think this is way too much detail for main text and authors run the risk of losing readers, for what is a very valuable SR.\n\nNow I have read further I realise you have a long section - starting on P14 – on the results – as they relate to IHC concepts. After having read that very long section, I still feel the need for some kind of punchy summary of the key findings (e.g. harms, COIs, costs, other options not often mentioned…) somewhere in the results section. This kind of summary doesn’t appear until start of Discussion – but I think could be included earlier.\n\nP18: I wonder if the dot points at bottom of P18 might be better in the Results – and help to drive the kind of summary I am suggesting above.\n\nDiscussion: the paper feels like it morphs in the Discussion into a passionate advocacy for IHC materials – with a sometimes moralistic tone. I think the tone could be addressed. And I also think that in order to justify the IHC material in the Discussion, more needs to be said about IHC in the Introduction – and the authors relations with IHC need to be made more explicit in the Methods – as noted above.\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? Partly", "responses": [ { "c_id": "7761", "date": "02 Feb 2022", "name": "Matt Oxman", "role": "Author Response", "response": "We are grateful to you and the other reviewers for your time, effort, and constructive criticism. Thank you! We have pasted your comments below, in italics, with responses in turn. In relation to the use of the word “qualitative” - I wonder if the authors might consider using a different word – or adding more explanation – perhaps saying “qualitative summary of results” – as it sounds to the casual reader like when you refer to “qualitative” you may have done a SR of “qualitative” studies of news coverage (i.e. focus groups with journalists etc.) – whereas I think you have a different meaning in mind. (e.g. P8 “qualitative summary”, which is also mentioned in Abstract). We have replaced “qualitative summary” with “review” in the abstract. And at first mention of “qualitative summary” in the main text, we explain that it is, not a synthesis of qualitative studies, but a structured synthesis of results excluded from the meta-analyses. Re: IHC. Given the importance of IHC concepts for this SR, and the role of the authors in IHC world, and the advocacy of IHC in the Discussion, I think the IHC concepts etc. needs to be mentioned in the Introduction and expanded on in the Methods (as to how and why exactly the IHC concepts interacted with your analysis in the SR) - and the authors link to IHC can be mentioned in Methods somewhere. This is important and will help explain how the Discussion section becomes a little like advocacy for IHC materials. We have added a paragraph about IHC to the end of the introduction, where we mention the IHC Key Concepts and the relevant authors’ involvement in the project. In the methods section, on pages six to seven in the first version of the article, we already explained why and how we used the IHC Key Concepts framework in the review. We do not think it is necessary to mention the authors’ involvement in IHC in the methods section. We have removed the last three paragraphs of the discussion, which were about IHC. The paper is long and complex – but with much valuable information. If any more material can be moved into Supp files, that will be helpful. We agree that the article is long and complex, which follows the study being large and complex. There are already 10 supplementary files and six supplementary tables, and we do not think it would be helpful to have more. We appreciate that the article was a demanding read for the reviewers, however we do not expect many other people will read it start-to-finish. The findings are summarised in the abstract and “Key findings” section, and the implications are summarised in Table 5. Moreover, there will be a short, plain-language post on the F1000Research blog summarising the findings and discussing the implications. Although we include implications for other groups, the target audience for the article itself is primarily researchers. Communicating the findings and implications to other groups is a separate task. Search strategy: It seems almost 2 years since it was updated. Authors might consider updating before indexing. It is possible that there have been new, eligible studies since the last search, and we agree that an updated review could be of value, at some point. Others planning or reporting new studies may, indeed, want to conduct updated searches. However, that is beyond the scope of this review, which summarises relevant research up to August 2019.There are good reasons for ensuring that reviews of interventions, which inform healthcare decisions are up to date when they are published (and are kept up to date). However, this is a review of descriptive studies and we do not believe those reasons are relevant to this review. P5: “We excluded inappropriate criteria…’ Perhaps “ineligible” might be better? We have replaced “ineligible [criteria]” with “inappropriate”, in the main text and Figure 1. P7: Re: your sentences “Furthermore, we reframed negative quality criteria as positive, and paraphrased all quality criteria, for the data to be consistently framed and worded, and for the criteria to be succinctly labelled. For example, Bubela et al. report the number of news reports that “did not” specify whether a study was randomized, which was reframed as the number of news reports that did specify whether a study was randomized.” While this makes sense, I presume that sometimes that re-framing was not straight forward – and I wonder if there is anything more to say on any complexities associated with this approach. Also, if you can give a little more detail in the Bubela example, with numbers, that could help the reader. We found reframing the criteria straightforward, and there were few criteria that needed to be reframed as positive. As noted in the main text, each criterion included in the meta-analyses is presented in S10 File verbatim, as reported in the primary study, together with the reframed and paraphrased version that we used in the review. We have added numbers to the example with the criterion from the study by Bubela et al. P7: “Risk of Bias”. Can the authors add a few words in text explaining why the \"risk of bias\" was solely about the risk of detection bias? A reader of an SR might normally expect a few different domains, so it would be good to just explain why this single domain was most relevant. We have added a paragraph about why we only assessed detection bias to the relevant section, with reference to the protocol and the section on deviations from the protocol, where we provide a detailed explanation. P10: Table 2. I think Table 2 needs re-thinking – as it took me some time to work out what was going on. I was treating each row as a discreet coherent entity, but in fact you have multiple parts to each row. I am sure you can make it clearer somehow. We have asked the journal to change the formatting of Table 2 so that the columns are alternate colours, rather than the rows. P11: “In contrast, a checklist for people communicating evidence-based information about the effects of healthcare interventions includes the item “Help your audience to avoid misinterpreting continuous outcome measures,” granted the checklist is not for journalists.” I don’t fully understand meaning of this sentence - perhaps be a little clearer? Mentioning the item in the checklist was meant to emphasise that the concept is important for laypeople. Upon reconsideration, we have simply removed the sentence since it was unclear and inessential. P12: This is a structural suggestion. By the sub-heading “Synthesis of Results” I am really wanting to hear some of the key findings – in terms of content (i.e. a strong compelling prose version of the data in Figure 3) – but I feel I am still getting a lot of complex fine detail about the characteristics of studies and elements of them. I understand there is a lot to report – but some clear statements of the key findings – even summarised and abstracted a little – would be helpful around here to orient the reader. We have added a summary to the start of the “Synthesis of results” section, by moving up content from the “Key findings” section, like you suggest further down. Also – a related point - I find the three paragraphs on P12 (and into P13) complex and hard to follow. I wonder whether this level of detail is necessary in the main text – and whether it might be moved to a supplementary – but that is for authors to decide. At the very least – if there is any way to make it all a little clearer around this point – that would be great for the reader. An example of the mention of the “ninth criteria” from Bubela and 12th from Haneef. I think this is way too much detail for main text and authors run the risk of losing readers, for what is a very valuable SR. Please see our response to your comment further up, about moving more material into supplementary files. Now I have read further I realise you have a long section - starting on P14 – on the results – as they relate to IHC concepts. After having read that very long section, I still feel the need for some kind of punchy summary of the key findings (e.g. harms, COIs, costs, other options not often mentioned…) somewhere in the results section. This kind of summary doesn’t appear until start of Discussion – but I think could be included earlier. P18: I wonder if the dot points at bottom of P18 might be better in the Results – and help to drive the kind of summary I am suggesting above. We have added a summary to the start of the “Synthesis of results” section, by moving up content from the “Key findings” section, including the dot points. Discussion: the paper feels like it morphs in the Discussion into a passionate advocacy for IHC materials – with a sometimes moralistic tone. I think the tone could be addressed. And I also think that in order to justify the IHC material in the Discussion, more needs to be said about IHC in the Introduction – and the authors relations with IHC need to be made more explicit in the Methods – as noted above. We have removed the last three paragraphs of the discussion, which were about IHC. Thank you again for the valuable feedback!" } ] }, { "id": "90763", "date": "04 Aug 2021", "name": "Joel R. Lexchin", "expertise": [ "Reviewer Expertise Pharmaceutical policy", "intellectual property rights" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 extremely comprehensive and well-done systematic review on an important topic. The authors have obviously put an enormous amount of effort into the project and the results show that. The study is well written and easy to follow. However, there are some areas where I believe that improvements can be made.\n\nThere is a potentially relevant article that the authors appear to have overlooked: Cassels et al. Open Medicine 2008;2(1):e20–23. Another study (Cassels et al., 2003) is a longer version of one that the authors use – reference 48.\n\nPage 3: The authors use a 2009 paper to claim that most people get health information either directly or indirectly from traditional news media as opposed to material that appears only on social media. However, studies that evaluate information sourced from the two different types of media may no longer be relevant to the present-day environment.\n\nPage 18: The authors say that their findings come from a diverse range of countries but don’t they mean primarily western countries?\n\nPage 20: The length of a newspaper story is not necessarily a measure of quality. Cassels et al. have shown that there are few significant differences in the overall quality between short news briefs and longer stories. (Cassels et al., 2003)\n\nPage 22: The recommendations for improving the reporting of journalists, editors and news outlets at least partially assume the existence of resources and time to educate journalists and editors about science journalism. However, the authors do not assess whether the necessary resources are actually present and available especially in smaller media outlets.\n\nPage 22: Informed Health is only available in English and German. Therefore, it is limited in terms of usefulness to those speaking other languages. Cochrane Evidence is superior in that regard.\n\nPage 23 (last line): Is it Informed Choices or Informed Health? Are these the same or different?\n\nPage 24: Earlier the authors refer to Informed Health and Informed Choices. Now there is a third name Informed Health Choices. Are all three the same?\n\nPage 24: Have any of the educational interventions that the authors mention (references 99-104) looked at the long-term (e.g., more than 1 year) effects?\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": "7762", "date": "02 Feb 2022", "name": "Matt Oxman", "role": "Author Response", "response": "We are grateful to you and the other reviewers for your time, effort, and constructive criticism. Thank you! We have pasted your comments below, in italics, with responses in turn. 1. There is a potentially relevant article that the authors appear to have overlooked: Cassels et al. Open Medicine 2008;2(1):e20–23. We excluded Cassels et al. 2008 based on the fourth eligibility criterion, specifically for inadequate specification of the selection criteria for news reports and missing specification of the selection technique. Another study (Cassels et al., 2003) is a longer version of one that the authors use – reference 48. We assume you are referring to the paper “Drugs in the News: How well do Canadian newspapers report the good, the bad and the ugly of new prescription drugs?” published by the Canadian Centre for Policy Alternatives. We were unaware of this version. However, it does not include data that we were missing from the shorter version. 2. Page 3: The authors use a 2009 paper to claim that most people get health information either directly or indirectly from traditional news media as opposed to material that appears only on social media. However, studies that evaluate information sourced from the two different types of media may no longer be relevant to the present-day environment. Our claim is that “people still get health information from the [news media],” not that most people get information directly or indirectly from traditional news media. We agree that distinguishing news and social media is challenging, and not necessarily sensible, which is an important point for new primary studies of the quality of health information in mass media. 3. Page 18: The authors say that their findings come from a diverse range of countries but don’t they mean primarily western countries? Yes, except we mean English-speaking and high-income countries, not Western. We have specified this. 4. Page 20: The length of a newspaper story is not necessarily a measure of quality. Cassels et al. have shown that there are few significant differences in the overall quality between short news briefs and longer stories. (Cassels et al., 2003) We agree and we have removed the second clause of the sentence, which was a possible explanation for the finding that features were generally of higher quality than news reports. 5. Page 22: The recommendations for improving the reporting of journalists, editors and news outlets at least partially assume the existence of resources and time to educate journalists and editors about science journalism. However, the authors do not assess whether the necessary resources are actually present and available especially in smaller media outlets. In the section “Implications for citizens and policymakers”, we now specify that news outlets can improve their reporting “with enough resources”. In the section “Implications for journalists, editors, and news outlets”, we had already specified that news outlets should provide or support training, and provide adequate time and resources, “if possible”. And in the section “Underlying problems”, we had already pointed out that in their synthesis, Amend and Secko found limited time or space for telling stories, and limited budgets and staff were barriers to high-quality health and science journalism. In fact, we assume most outlets either cannot provide training or resources to improve their health and science journalism, or will not prioritise such spending, which is part of why we argue that it is so important to help all people, not just journalists, learn how to think critically about health information for themselves. That said, we already referred to free learning resources that journalists can use to improve their skills, without necessarily spending a lot of time. 6. Page 22: Informed Health is only available in English and German. Therefore, it is limited in terms of usefulness to those speaking other languages. Cochrane Evidence is superior in that regard. We agree, but it is still helpful to be aware of both sites. 7. Page 23 (last line): Is it Informed Choices or Informed Health? Are these the same or different? It is “Informed Health”. We have corrected the typo. 8. Page 24: Earlier the authors refer to Informed Health and Informed Choices. Now there is a third name Informed Health Choices. Are all three the same? At first mention of the Informed Health website, we now note that it is a separate initiative from the Informed Health Choices (IHC) project. Again, where we have referred to the site as “Informed Choices”, it should have said “Informed Health”, and we have fixed this. 9. Page 24: Have any of the educational interventions that the authors mention (references 99-104) looked at the long-term (e.g., more than 1 year) effects? No, and we believe that a series of interventions is necessary, reinforcing and building on what people have learned. However, this has not been tested. Note that we have removed the last three paragraphs, in response to Review 1. Thank you again for the valuable feedback!" } ] }, { "id": "90288", "date": "09 Aug 2021", "name": "Amanda Wilson", "expertise": [ "Reviewer Expertise Public health", "health literacy", "health reporting in the media", "health education" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a systematic review of studies assessing the quality of health news articles which provides a meta-analysis of the results. This is an interesting paper which provides a quantitative approach to what is likely to be considered a difficult area to quantify – that is quality of reporting. The influence of news reporting on health behaviours is well known and the current climate of pandemic and vaccination information makes this a highly pertinent area to explore.\nThe methodology is sound and rigorous with the authors following the gold standard approach of registering the review and publishing the protocol. All steps of the literature review are described in detail and the analyses and interpretation are sound.\nThe paper is extremely well written and I have little critical feedback on the structure of the research or presentation of the paper. My only criticism is that it is extremely long and possibly overly detailed. The length and detail are worthy of a Cochrane review but without the saving grace of a plain language summary. I wonder if some ruthless editing might enhance the number of readers who make it the whole way through. It feels like a PhD chapter rather than a journal article. Having said that, in the Discussion on page 18, I was interested to know which criteria did not make sense and which aspects of quality were not covered.\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": "7763", "date": "02 Feb 2022", "name": "Matt Oxman", "role": "Author Response", "response": "We are grateful to you and the other reviewers for your time, effort, and constructive criticism. Thank you! We have pasted your comments below, in italics, with responses in turn. My only criticism is that it is extremely long and possibly overly detailed. The length and detail are worthy of a Cochrane review but without the saving grace of a plain language summary. I wonder if some ruthless editing might enhance the number of readers who make it the whole way through. It feels like a PhD chapter rather than a journal article. Please see our response to Review 1. Having said that, in the Discussion on page 18, I was interested to know which criteria did not make sense and which aspects of quality were not covered. Regarding criteria that did not make sense, some conflict with IHC Key Concepts, such as the example in Table 3, or criteria that conflate a “statistically significant” effect with an important effect, including respective criteria used by Haneef et al. and Yavchitz et al. Others are imprecise measures of quality, for example criteria focusing on the novelty of the health intervention, or how much of a news report was based on a press release, since a report can be low or high-quality regardless of these characteristics. Regarding the aspects of quality not covered, S6 Table shows which IHC Key Concepts were not related to any criteria included in the meta-analyses. Thank you again for the valuable feedback!" } ] }, { "id": "90284", "date": "23 Aug 2021", "name": "David Henry", "expertise": [ "Reviewer Expertise clinical epidemiology", "pharmacoepidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe other reviewers of this article have highlighted its considerable strengths and I won’t repeat their comments. This is a solid attempt to summarise studies that have made structured attempts to quantify and report key characteristics of medical news stories that report therapeutic claims. It is very useful to have this summary.\nMany of these studies were carried out to highlight the weaknesses in the reporting of medical advances in the mainstream media in the hope that this would raise standards. But there is no strong evidence that this has resulted from these efforts.\nThe studies included in this review preceded the news coverage of COVID-19, its preventive and therapeutic interventions. It would be very instructive to measure how the media performed during the epidemic and whether reporting evolved. My informal impression is that respect for science, and possibly the reports themselves, have improved. However, this has been countered by ignorant and some willfully and politically motivated inaccurate reporting in a few mainstream outlets and social media. My point is that the media landscape may have changed because of Covid-19.\nI only have a few specific comments:\nI don’t think that conventional meta-analysis produces meaningful data in this situation. Meta-analysis is at its best when we can assume that there is an underlying single effect of interest that is measured imprecisely by individual studies. For instance, the relative effects of many important medications (e.g, statins, anticoagulants), which seem constant across different settings and background risks. Meta-estimates of prevalence are less helpful as there is usually an assumed variation. That is the case here where the study methods, story topics, settings, and reporters vary. There is no reason to assume a common effect to be meta-analysed. Some of the I2 values are huge. In my view it would be preferable to present more as a scoping review, displaying and discussing the range of values reported by the studies.\n\nThe investigators, appropriately, have mapped the varying scales and items to a common framework. They have used their own Informed Health Choices (IHC) Key Concepts framework. That’s fine, but the paper seems to assume a familiarity with the framework that many readers will not possess. I think a simplified version of the framework needs to be presented in the main text. At present, it seems to be buried in Supplementary data.\n\nIn the Abstract, the authors conclude: ‘There is room for improving health news, but it is logically more important to improve the public’s ability to critically appraise health information and make judgements for themselves.’ The paper does not provide data to support this conclusion. The authors have done important ground-breaking work with IHC but the extensive coverage they provide themselves in the Discussion of an already long paper seems excessive. A separate editorial/opinion piece making these points would be welcome.\n\nI think there may be a mistake in the dot point summary in the Discussion which states ‘• 86% of news reports commit “disease mongering” (95% CI 78%-91%) (low certainty).’ In the paper they state ‘Overall, 86% of news reports do not commit disease mongering (95% CI 78%-91%)’. I think the latter is correct.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "7764", "date": "02 Feb 2022", "name": "Matt Oxman", "role": "Author Response", "response": "We are grateful to you and the other reviewers for your time, effort, and constructive criticism. Thank you! We have pasted your comments below, in italics, with responses in turn. 1. I don’t think that conventional meta-analysis produces meaningful data in this situation. Meta-analysis is at its best when we can assume that there is an underlying single effect of interest that is measured imprecisely by individual studies. For instance, the relative effects of many important medications (e.g, statins, anticoagulants), which seem constant across different settings and background risks. Meta-estimates of prevalence are less helpful as there is usually an assumed variation. That is the case here where the study methods, story topics, settings, and reporters vary. There is no reason to assume a common effect to be meta-analysed. Some of the I2 values are huge. In my view it would be preferable to present more as a scoping review, displaying and discussing the range of values reported by the studies. We assume that this comment is suggesting the studies were too heterogeneous for estimating average proportions across studies to be meaningful. We were conservative about synthesising results this way, and cautious in our interpretation. We used a random effects analysis, which explicitly assumes there is not a common effect, but rather that the studies estimate distinct effect sizes. The large I2 values are a property of the literature included. The range and results for individual studies are available in the supplementary material, with the forest plots. We elected to exclude this information from the text because it would make the text harder to read, and it would not substantially alter the results—i.e., despite substantial heterogeneity, the overall estimates and confidence intervals provide an appropriate indication of what was found across the studies in each meta-analysis, and a better indication than the median and range, or range alone. 2. The investigators, appropriately, have mapped the varying scales and items to a common framework. They have used their own Informed Health Choices (IHC) Key Concepts framework. That’s fine, but the paper seems to assume a familiarity with the framework that many readers will not possess. I think a simplified version of the framework needs to be presented in the main text. At present, it seems to be buried in Supplementary data. The article is already long, as pointed out in Reviews 1 and 3. We have included the framework as S6 File and reference the file at first mention of the framework in the methods section, where we also summarise its content, and explain why and how we used it. 3. In the Abstract, the authors conclude: ‘There is room for improving health news, but it is logically more important to improve the public’s ability to critically appraise health information and make judgements for themselves.’ The paper does not provide data to support this conclusion. The authors have done important ground-breaking work with IHC but the extensive coverage they provide themselves in the Discussion of an already long paper seems excessive. A separate editorial/opinion piece making these points would be welcome. We have removed the last three paragraphs of the discussion, which were about IHC. 4. I think there may be a mistake in the dot point summary in the Discussion which states ‘• 86% of news reports commit “disease mongering” (95% CI 78%-91%) (low certainty).’ In the paper they state ‘Overall, 86% of news reports do not commit disease mongering (95% CI 78%-91%)’. I think the latter is correct. Correct. We have fixed this typo. Thank you again for the valuable feedback!" } ] } ]	1	https://f1000research.com/articles/10-433
https://f1000research.com/articles/10-795/v1	11 Aug 21	{ "type": "Research Article", "title": "Association between traumatic brain injury (TBI) patterns and mortality: a retrospective case-control study", "authors": [ "Gilbert Koome", "Faith Thuita", "Thaddaeus Egondi", "Martin Atela", "Faith Thuita", "Thaddaeus Egondi", "Martin Atela" ], "abstract": "Background: Low and medium income countries (LMICs) such as Kenya experience nearly three times more cases of traumatic brain injury (TBI) compared to high income countries (HICs). This is primarily exacerbated by weak health systems especially at the pre-hospital care level. Generating local empirical evidence on TBI patterns and its influence on patient mortality outcomes is fundamental in informing the design of trauma-specific emergency medical service (EMS) interventions at the pre-hospital care level. This study determines the influence of TBI patterns and mortality. Methods: This was a case-control study with a sample of 316 TBI patients. Data was abstracted from medical records for the period of January 2017 to March 2019 in three tertiary trauma care facilities in Kenya. Logistic regression was used to assess influence of trauma patterns on TBI mortality, controlling for patient characteristics and other potential confounders. Results: The majority of patients were aged below 40 years (73%) and were male (85%). Road traffic injuries (RTIs) comprised 58% of all forms of trauma. Blunt trauma comprised 71% of the injuries. Trauma mechanism was the only trauma pattern significantly associated with TBI mortality. The risk of dying for patients sustaining RTIs was 2.83 times more likely compared to non-RTI patients [odds ratio (OR) 2.83, 95% confidence interval (CI) 1.62-4.93, p=0.001]. The type of transfer to hospital was also significantly associated with mortality outcome, with a public hospital having a two times higher risk of death compared to a private hospital [OR 2.18 95%CI 1.21-3.94, p<0.009]. Conclusion: Trauma mechanism (RTI vs non-RTI) and type of tertiary facility patients are transferred to (public vs private) are key factors influencing TBI mortality burden. Strengthening local EMS trauma response systems targeting RTIs augmented by adequately resourced and equipped public facilities to provide quality lifesaving interventions can reduce the burden of TBIs.", "keywords": [ "Trauma Patterns", "Patient Characteristics", "pre-hospital Care", "Traumatic Brain Injuries", "Trauma mortality" ], "content": "Abbreviations\n\nAOR, adjusted odds ratio; CI, confidential interval; CNS, central nervous system; EMS, emergency medical services; GCS, Glasgow Coma Scale; LMIC, low- and medium-income country; RTI, road traffic injury; SE, standard error; TBI, traumatic brain injury; TI, traumatic injury.\n\n\nIntroduction\n\nTrauma is a serious global public health problem.1 Traumatic injuries (TIs) are estimated to account for 10% of all deaths and about 5.8 million deaths annually and at least 6% of Years Lived with Disability (YLD).2,3 Low and medium-income countries (LMICs) account for about 90% of this global trauma disease burden.4 Globally, traumatic brain injury (TBI) is the leading form of TI burden.1 Currently, about 69 million people suffer from TBI annually, mainly from road traffic injuries (RTIs), violence and falls.7,8 The estimated economic cost of RTIs in Europe is substantially high with an approximate range of 7500-1,200,00 US dollars.5 Young persons aged less than 40 years are the most affected.6 In the EU, over 1.5 million people are admitted to hospital for TBI annually, with Austria and Germany reporting about eight times more admissions compared to Portugal and Spain. Of these admissions, about 50,000 deaths, 230,000 hospital admissions and 1.1 million discharges are reported.7 One study found EU hospital admissions, adjusted for population, to be three times higher compared to the USA.7 This indicates significant inter-continental TBI burden and trauma system development variations.\n\nLMICs, mainly in Africa, experience about three times more cases of TBIs compared to high income countries (HICs) such as EU and USA.8 This constitutes approximately 80% of the TBI global burden, most cases of which are potentially preventable using quality and effective pre-hospital care emergency medical service (EMS) systems.4,9 In these countries, TBI remains a growing public health burden concern. There are increasing concerns that EMS at the pre-hospital care level is ineffective and incapable of adequately mitigating increasing number of TBIs requiring critical care interventions.10 The economic cost of TBIs, such as mortality, morbidity and high hospital bills, has serious economic impact at an individual, household and societal level.11 For instance, LMICs, mainly in sub-Saharan Africa, lose about four billion United State dollars (US$) annually due to RTIs, a major cause of TBIs. This is equivalent to 11% of the their gross domestic product (GDP).10,12 In Kenya, the cost of RTIs is estimated at 14 billion Kenya shillings per year.13\n\nIn Kenya and other LMICs, the greatest proportion of TBI mortality and morbidity burden is attributed to poor access to quality emergency care.14 In HICs such as America and Europe, about half of such preventable mortality is said to occur at the pre-hospital care level.15 The proportion and impact is estimated to be three times as high in LMICs.8 The disproportionately high burden and cost of TBI in Africa and other LMICs are exacerbated by weak health systems, especially at the pre-hospital care level, and limited reliable empirical evidence to inform effective response to the growing trauma burden.4,16–19\n\nIdeally, TBI patients are expected to receive quality pre-hospital care, also known as “life-saving interventions” from trained and qualified health professionals before pre-hospital transport and/or reaching a specialized trauma care facility, also know as Tertiary Facilities. However, few patients receive this care, with most victims receiving no life-saving interventions due to lack of qualified staff and resourced EMS facilities at this care level.14 Evidence affirms a lack of a well-coordinated and integrated pre-hospital trauma care system in these settings to respond to growing TBI burden.20 Thompson et al.21 described Kenya's EMS for pre-hospital trauma care such as TBIs as fragmented, poorly coordinated and ineffective. Weakness in the health system has been linked to, among others, inadequate resources, staff, leadership, lack of training standard, lack of emergency trauma specialists, lack of effective communication systems and ineffective EMS response systems.22–24 In Kenya and other LMICs, evidence to support the development of local and adaptive life-saving interventions for averting or reducing growing TBI mortality at pre-hospital care level are grossly lacking.11,25\n\nTBI patterns comprise a complementary component of a responsive trauma/injury assessment and response at the pre-hospital care level. For instance, effective EMS response may require responses matched to the specific trauma source, injury type and its severity level.26 In this study, trauma pattern is defined in three different ways; based on source (RTIs and non-RTI), type of injury (blunt and penetrating) and day of injury (weekday and weekend). Reviews indicate the main cause of TBIs is RTIs, followed by violence and falls.10,22,27 TBI burden from RTIs has continued to exert pressure on already weak health systems globally due to increasing motorization and lack of effective EMS response systems.28–31 For instance, worldwide, about 1.2 million RTIs occurred in 2012, with male persons aged 15-29 years being the most affected.32 In Ghana, about 50% of TIs (TBIs included) are reportedly traffic and falls-related.22,27 In Kenya, more than 75% of health facilities’ emergency department visits are also due to RTIs.12 In LMICs alone, RTIs are estimated to increase by about 80% in the next decade due to an increase in motorization, low adherence to road traffic regulations and lack of effective EMS at the pre-hospital care level.10\n\nSource of trauma has been associated with increasing mortality burden from all trauma forms - RTIs have been linked to increasing burden of all forms of trauma mortality33–35 but no similar findings on TBI mortality have been found. Limited evidence available indicates that penetrating trauma or injuries are associated with a significantly longer length of stay in hospital.28 Penetrating trauma has also been found to increase all forms of trauma mortality significantly.36,37 Blunt trauma has been closely linked to motor vehicle collisions and falls.38 No study was found to examine the relationship between TBI type and mortality in LMICs. Access to prompt and quality care, at both the pre-hospital and in hospital level, can vary substantially by day of injury. Availability of trained staff and health services is higher during weekdays compared to weekends.39,40 For instance, during weekends, there could be fewer trained providers to promptly respond to EMS needs. Available studies focus mainly on injury admission day - which may be different from injury day considering long pre-hospital delays in Kenya and other LMICs - and are mainly descriptive.19,28,39 These studies report higher prevalence of TIs during weekends than on weekdays due to high trauma risk activities such as social events and mobility. According to a South African study by Möller et al., hospital admission day is significantly associated with all forms of trauma mortality.36 However, studies examining the influence of TBI day on mortality could not be found in the literature.\n\nIn Kenya and other low resource countries, preventive public health measures such as road safety measures and laws like using motorcycle helmets and observing road traffic regulations, have been instituted to reduce trauma burden and address some of the identified EMS response weaknesses. These measures seem to have failed to substantially reduce mortality and disability arising from TBIs and other TIs.17 Understanding TBI patterns and their influence on mortality can provide important insight on occurrence, presentation, diagnosis and alternative interventions for improving patient survival outcomes. Characterizing these patterns can enable EMS and other health professionals to understand and mitigate presentation of TBIs and possible risk factors for avoidable mortality.30,41 The evidence can also offer critical insight in designing locally adaptive and effective responses for the pre-hospital trauma care level, yet this evidence is widely lacking in locally published literature. It is with this background that this study aimed to test the hypothesis that the patterns of TBI was not associated with mortality at the pre-hospital care level.\n\n\nMethods\n\nWe conducted a retrospective unmatched case-control study through data abstraction from hospital-based patient records. This study was done in three leading tertiary referral facilities comprising public (Kenyatta National Hospital, KNH) and private (Kikuyu Mission and Mater Misericordiae Hospital) tertiary trauma facilities in Kenya. Data was abstracted from 316 TBI patient medical records (consisting of 158 cases and 158 controls) for the period of January 2017 to March 2019 in the three hospitals. The study sample was calculated using Kelsey’s unmatched case control formula.42 The formula assumptions were as follows, proportion of cases exposed; 0.667, proportion of controls exposed; 0.6 and case-control ratio of 1.0. Cases were patients who had died within 30 days after the trauma while controls were patients who survived for at least 30 days after trauma. At the facility, all TBI patients meeting the inclusion criteria were sampled and listed. Patients with TBIs were identified using International Classification of Diseases (ICD) codes assigned to patient medical records or files. A separate list of cases and controls which met the study inclusion criteria was developed. In situations where more than one control was identified or available, simple random sampling using a random number generator was used to sample one control from the list. Controls for cases were derived from the same facility to mitigate bias associated with difference in quality of care in different facilities.\n\nIn the study, patients with TBIs based on ICD diagnosis codes received in the three selected trauma referral hospitals for the period between January 2017 to March 2019 were included. Immediate death on the scene and patients not transferred to hospitals due to minor injuries were excluded from the study. Immediate deaths on injury scene due to severe injuries presents little opportunity for improving care through life-saving interventions while minor injuries such as bruises presents no major mortality risks.\n\nDuring sampling, immediate cases/deaths occurring at the injury scene were excluded due to inability to obtain comprehensive data and the minimal opportunity for providing pre-hospital care or life-saving interventions. Further, patients admitted in lower level facilities for more than 24 hours after injury were excluded due to potential of confounding linked to differences in care quality in the two levels of care delivery, that is, lower and tertiary level facilities. Pediatric trauma may require different critical care response compared to adult trauma, hence exclusion of patients aged less than 18 years to avoid response-specific bias. Patient records with missing information of at least 5% of the abstracted data were also not included in the analysis.\n\nData abstracted included mortality outcomes, that is: whether the patient died or survived; trauma patterns comprising day of injury (Monday/Tuesday/Wednesday/Thursday/Friday/Saturday/Sunday), type of injury (penetrating/blunt) and injury mechanism (RTI/non-RTI such as violence, falls and gunshots); demographic characteristics comprising age (18-29 years/30-39 years/40-49 years/50-59 years/60+ years) and gender (male/female) and vital patient characteristics consisting of Glasgow Coma Scale (GCS) score (severe, moderate and mild), presence of hypoxemia, defined as blood oxygen concentration of less than 90%, (Yes/No), presence of comorbidity (Yes/No), alcohol involvement (Yes/No), patient triage status (not urgent/urgent/emergency) and blood pressure levels (hypertension/elevated/normal). We also collected data on access to pre-hospital care categorized as Yes/No. To improve completeness and accuracy, abstracted data were complemented with other pre-hospital care records including ambulance records, referral notes and trauma registries maintained at the Accident and Emergency Departments. For deceased patients, mortality and death notification reports were used to collate information on death which included cause of death, place of death and the patient demographics comprised of age and gender.\n\nFor descriptive analysis, we performed bivariate analysis to assess mortality outcome differences among cases and controls, including both individual and trauma pattern characteristics. We used Pearson’s chi-square test to assess differences in proportions and Student’s t-tests to assess differences in mean patient ages. Logistic regression was used to assess association between mortality and trauma patterns adjusting for other predictor variables. To identify possible confounding effects, existence of a 10% difference between unadjusted and unadjusted regression coefficient was used.43,44 Statistical significance at 0.05 level between exposure and outcome was also taken into account.43 Using these statistical methods, type of transfer tertiary facility and trauma severity score (GCS) were found to confound TBI mortality outcome, hence they were included in the analysis as control variables. Access to life-saving interventions at the pre-hospital care level may result in lower mortality risks for the different trauma patterns. Access to pre-hospital care was defined as the provision of life-saving interventions by a qualified health professional such as a paramedic, nurse, clinical officer or medical officer. To account for this possible confounding effect in outcome, we included access to pre-hospital care as an important control variable in the model. Based on previous studies and other published literature, we also selected patient and injury characteristics that were clinically and substantively (statistically significant at bivariate analysis level) relevant to be included in the adjusted logistic regression model. In this paper, we report the adjusted odds ratio (AOR) for mortality, after controlling for age, gender, trauma severity, presence of hypoxemia, presence of comorbidity, pre-hospital time, type of transfer tertiary facility and access to pre-hospital care. Due to small sample size in some of the age and injury day variable categories, some of the categories were combined in the regression model - age was re-constituted to three categories (18-29 years/30-39 years/40+ years and injury day to two categories (weekday and weekend). Abstracted medical data was analyzed using IBM SPSS statistics software, version 26. A p-value of less than 0.05 was considered statistically significant.\n\nSince the study involved de-identified retrospective data abstracted from many patient records - some of whom were deceased - it was difficult to reach and contact all the respondents and obtain informed consent, particularly, the deceased. A waiver of consent for data abstraction was granted by the Kenyatta National Hospital- and University of Nairobi Ethics and Research Committee (KNH-UoN/ERC/FORM/IC05). A research permit was obtained from the National Commission for Science, Technology and Innovation (NACOSTI/P/19/9613/31326). Institutional ethical clearance was also obtained from all the three hospital ethical boards prior to data collection. There is no identification or individual details presented in this article or data thereof. This is in line with the waiver for consent obtained during ethical approval of this study.\n\n\nResults\n\nA descriptive summary of patient characteristics and mortality outcomes is shown in Table 1. The mean age of patients was 34.5 years and there was no significant difference between cases and controls. Eighty-five percent of the patients were males. The distribution of gender was similar in both cases and controls (82% versus 88%). The distribution of blood pressure levels (hypertension, elevated and normal) was the same among cases compared to controls. The number of severely injured patients was significantly higher among cases compared to controls (65% versus 34%). Similarly, the number of patients triaged as non-urgent was significantly higher among cases than controls (30% versus 22%). However, the number of patients triaged as emergency cases was significantly higher in controls compared to cases (47% versus 41%).\n\nStatistical significance (Probability(P) values) is shown in asterisks. Number of cases are 158, Controls are 158 and total population are 316 persons. In the table, + means “and above”, “<” means less than and “>” means more than. Parenthesis (-) shows range of values while % means percentage.\n\n* p≤0.05.\n\n*** p≤0.001.\n\nHypoxemia was present in 31% of the patients. The number of hypoxemic patients was significantly higher in cases compared to controls (25% versus 38%). Turning to comorbidity, 39% of the patients were diagnosed with comorbidity. The distribution of comorbidity was similar among cases and controls (42% versus 36%). The pattern was different among 28% of the patients who had a history of alcohol use. The distribution of patients with a history of alcohol use was significantly higher among cases compared to controls (33% versus 22%). In respect to pre-hospital time, 57% of patients arrived at the tertiary hospital less than three hours after injury. Distribution of pre-hospital time was significantly different in both cases and controls. There were more patients arriving at the tertiary hospital in less than three hours among controls compared to cases (64% versus 50%). Injured patients are transferred from scenes to different tertiary trauma care hospitals for specialized care and management;53% of patients were transferred to a public tertiary hospital. The number of patients transferred to a public facility was significantly higher among cases compared to controls (63% versus 42%). Around half (56%) of patients received pre-hospital care. The number of patients who accessed pre-hospital care was significantly higher among controls compared to cases (63% versus 49%).\n\nWe examined three types of trauma patterns; trauma mechanisms (RTI and non-RTI causes), type of injury (blunt and penetrating) and injury day (weekday and weekend). A descriptive summary of trauma patterns by mortality outcomes is shown in Table 2.\n\nStatistical significance (Probability(P) values) is shown in asterisks. Number of cases are 158, Controls are 158 and total population are 316 persons. In the table, Parenthesis (-) shows range of values while % means percentage.\n\n* p≤0.05.\n\n*** p≤0.001.\n\nTrauma mechanisms\n\nRTIs were the main cause of TBIs (58%) compared to non-RTI causes (42%) which consisted of falls, violence and gunshots (Figure 1). RTIs and gunshots were the main source of trauma among patients aged 18-29 years, while violence was main source of trauma among patients aged 18-39 years. Motor vehicles (61%) were the main cause of RTIs compared to motorcycles. Trauma caused by gunshots, falls and violence was mainly reported in public places (54%), followed by home (27%) and workplace (18%). TBIs due to RTIs were significantly higher among cases compared to controls (67% versus 49%).\n\nRTI, road traffic injury.\n\nSource: Author.\n\nType of trauma or injury\n\nBlunt trauma (71%) was the main form of injury across all forms of trauma. Blunt trauma was mainly caused by RTIs and falls, while penetrating trauma was mainly caused by gunshots and violence-inflicted injuries. The most commonly injured body part was the head (89%) followed by lower extremities as shown in Figure 2. Record review indicated that concussions and contusions were the main form of head injuries attributed to TBIs. Skull fractures and scalp wounds were also frequently reported. Distribution of blunt trauma was significantly higher among cases compared to controls (77% versus 65%).\n\nInjury day\n\nCumulatively, 72% of injuries were recorded during weekdays, while 28% were recorded during weekends. The weekday has five days, Monday to Friday, while the weekend has only two days, Saturday and Sunday. The number of injuries was slightly higher on Friday compared to other weekdays. However, distribution of injuries was the same across all weekdays.\n\nWe performed logistic regression analysis (53) to examine association between trauma patterns and TBI mortality. Logistic regression results are shown in Table 3. Trauma mechanism was found to be significantly associated with TBI mortality both independently and after adjusting for other variables. RTI patients were 2.83 times more at risk of dying compared to non-RTI patients. Type of injury was found to be significantly associated with TBI mortality independently but this association became insignificant after adjusting for other variables. The risk of dying from blunt trauma was found to be 1.22 times higher compared to penetrating trauma. Further, injury day was not found to be significantly associated with TBI mortality both independently and after adjusting for other variables but the risk of TBI mortality was found to be 0.69 times lower in weekday injuries compared to weekend injuries.\n\nStatistical significance (Probability(P) values) is shown in asterisks. Number of cases are 158, Controls are 158 and total population are 316 persons. In the table, + mean “and above”, “<” means less than & “>” means more than. Parenthesis (-) shows range of values while % means percentage.\n\n* p≤0.05.\n\n p≤0.01.\n\n* p≤0.001.\n\nAt the individual level, patient age was not found to be significantly associated with TBI mortality even after adjusting for other variables but the risk of dying was found to be 0.92 times lower among younger patients aged 18-29 years compared to older patient aged 40+ years. Female gender was found to be significantly associated with TBI mortality after adjusting for other variables but not independently. A female patient was 1.64 times more at risk of dying compared to a male patient. Trauma severity was also found to be significantly associated with TBI mortality both independently and after adjusting for other variables. The risk of dying from a severe trauma (GCS<9) was 3.42 times more likely compared to mild or minor trauma (GCS 13-15). After adjusting for other variables, a hypoxemic patient was found to be significantly associated with a 4.75 times higher risk of dying compared to a non-hypoxemic patient.\n\nPresence of comorbidity was significantly associated with TBI mortality after adjusting for other variables but not independently. The risk of dying was 1.82 times more likely in comorbid patients compared to non-comorbid patients. Alcohol use was also significantly associated with TBI mortality both independently and after adjusting for other variables. Patients with a history of alcohol use were 2.57 times more at risk of dying compared to those with no alcohol use history. Pre-hospital time was not found to be significantly associated with TBI mortality after adjusting for other study variables but shorter pre-hospital transport time seemed to reduce the risk of dying. In respect to type of transfer tertiary facility, type of facility was found to be significantly associated with TBI mortality both independently and after adjusting for other variables. Transfer of patients to a public facility was associated with a 2.18 times higher times risk of dying compared to a private facility. Access to pre-hospital care was not found to be significantly associated with TBI mortality after adjusting for other variables but was found to lower TBI mortality risk by 0.58.\n\n\nDiscussion\n\nThis study sought to assess the association between trauma patterns and TBI mortality. We found one trauma mechanism in particular, RTIs, to be associated with a higher risk of TBI mortality. Trauma mechanism, mainly TRIs, has been previously studied and associated with all forms of trauma mortality.33–35 However, these studies did not adjust for access to pre-hospital care and type of trauma care facility, which are shown to be important confounders of mortality outcome. Globally, and particularly in sub-Saharan Africa, RTI is a major public health problem contributing to a high burden of post-trauma mortality and morbidity.28,33,34,36 More than 50% of trauma is RTI-related.22 In Africa and other LMICs such as Ghana, India, Kenya and Uganda, the negative impact of an increase in motorcycle injuries is an increasing public health problem reflected by huge cases of RTI-related fatalities and morbidities.22,28,33,34 Kenya has seen an unprecedented increase in motorcycle motorization, which has exponentially increased trauma burden such as mortality and disability.33 In some LMICs, injury burden from motorcycles has exceeded those from other motorized vehicles.28,33 This has been worsened by lack of EMS care capacity at the pre-hospital care level to respond to increasing demand for quality care in settings outside of hospital.45\n\nPedestrians, unrestrained passengers and motorists are the most commonly injured patients due to high exposure risks when crossing roads and walking along major roads. In addition to non-adherence to road traffic rules among motorists, poor road infrastructure contributes to increased risk of RTIs.16,22 For instance, many major roads have limited provisions for safe walkways and cycling paths, further exposing road users to avoidable injuries. Road safety and awareness campaigns have not been adequately successful in injury and mortality risk reduction.46,47 Complementing these efforts with effective on-scene EMS responses can provide possible survival and health outcome benefits. This requires continued mapping and review of trauma pattern profiles as well as their effect on mortality in both rural and urban areas to inform trauma-specific responses. Consistent with other studies,28 the head was found to be the most injured body part. This is expected because our study population are TBI patients. Chalya et al.28 reported head injuries are the leading cause of deaths and disability in TBIs. Head injuries are associated with an increase in odds of death of around 1.5 times48 compared to non-head injuries. Similar to this study, lower extremities were also found to be frequently injured. The limbs sustain wounds, abrasions laceration, amputations or fractures which constitute significant morbidity risks such as disability as well as deaths due to uncontrolled hemorrhage. Serious head injuries present with life-threatening outcomes such as intercranial hemorrhage, which require timely life-saving interventions. Brain contusion and concussions were frequent form of injuries attributed to blunt injuries. These brain injuries can pose significant mortality risks if not managed effectively and promptly in line with the \"Golden Hour\" concept.33,45 This concept argues that life-saving interventions are be provided within one hour after injury for high efficacy while preventing irreversible pharmacological changes associated with higher mortality risks. Internal head and brain injuries are easily missed (missed injuries) due to poor diagnosing capability and lack of trained staff at the pre-hospital care level. Gaps in pre-hospital life-saving responses significantly increase the risk of preventable mortality.33,45 Quality pre-hospital trauma responses matching skill and resource needs are lacking in these settings, hence a higher case load of avoidable fatalities and disabilities. This burden can be averted using effective and timely out of hospital settings EMS responses targeting this type of trauma.\n\nBlunt trauma is frequently reported in RTIs37,38 while penetrating trauma is frequent in gunshots, stabs and other invasive trauma. Type of injury (blunt or penetrating) was not a predictor of TBI mortality after adjusting for other variables and confounders. This is inconsistent with other studies which showed penetrating trauma to increase mortality significantly.36,37 However, unlike our study, in these studies, which included other types of trauma such as central nervous system (CNS) injuries and amputations, penetrating trauma was most frequently reported. This variation in trauma mechanism and pattern may partially explain the difference in results. Blunt trauma was associated with increased odds of mortality, although this association was not significant. This is attributed to the role of RTI which is the main source of blunt injuries.\n\nWe further investigated the effect of injury day on trauma mortality. We found distribution of injury to be similar across weekdays. There was also no significant difference in TBI outcomes when weekend and weekday injuries were compared. While the study doesn’t provide sufficient evidence to refute the existing argument that TI (morbidity) is a predominantly a weekend problem due to high number of social events and mobility,19,28,39 it suggests that TBI morbidity may not be predominantly a weekend problem as expected. We found no published study examining the influence of injury day on trauma mortality outcomes. One study by Möller et al. examined a different but related aspect, the influence of hospital admission day on all forms of trauma mortality.36 The study showed high trauma case admissions during weekends, especially between midnight and six o’clock in the morning compared to weekdays.36 In the study, hospital admission day was significantly associated with mortality in which more deaths were noticeable on Tuesdays and Fridays. However, due to the small sample size in which the observed cell frequencies for these days was less than five, generalization of the evidence was low.\n\nFurther, a descriptive study from Tanzania found higher rates of injuries during the day but did not examine the influence of day of injury dynamics on mortality.28 We note that during the day, injury exposure is high due to higher mobility due to travel to workplaces, engagement in economic activities and other social events. Our study did not examine the difference between day and night injuries due to a lack of data on this injury characteristic. A lack of robust and comprehensive trauma registries across LMIC countries that capture injury day and other injury data is the main reasons for this study gap. As a result, most authors use trauma admission time, which is easily found in patient records, as proxy indicator of injury day. In LMICs, pre-hospital delays can be as high as seven days due to missed injuries and under-triaging and hence are a possible reason for the difference in mortality outcome between injury day and admission day after trauma.\n\nWe also included type of transfer facility in the adjusted predictor model. To our knowledge, this is the first published study to control for type of tertiary facility as an important contextual and control factor that can significantly alter trauma mortality outcomes. We found type of tertiary transfer facility to be significantly associated with TBI mortality, both independently and after adjustment of other variables. In this study, risk of dying was significantly higher among patients transferred to a public tertiary facility compared to a private facility. Access to pre-hospital care (from trained EMS providers) and differences in care among public and private transfer facilities can alter post-trauma outcomes due to differences in quality of life-saving and trauma care interventions provided as part of care continuity. Patient transfer to well-equipped facilities can have substantive benefits for survival outcomes. In LMICs, private facilities are known to be better equipped and staffed than public facilities, hence creating variations in care quality. For instance, at minimum, a well-equipped trauma care facility should provide computerized tomography (CT) scanning, hemorrhage control, provision of IVs, neurosurgical care, Intensive Care Unit, intracranial monitoring and treatment among others as part of critical care.49 Availability of these facilities, resources and equipment may vary substantively across public and private facilities.\n\nIn Kenya and other LMICs, most public referral facilities have limited resources to attend to a high number of patients linked to poor leadership and underfunding, hence possible suboptimal care.9,17 There are also care delays due to over-triaging and ineffective governance systems to support timely and optimal trauma care compared to public facilities.14,26 In public facilities, there are limited theatres and experts to serve a high number of casualties especially in cases of mass casualty.49 Qualified trauma specialists are also frequently away in public practice, leading to delays in life-saving care. In private tertiary facilities, trauma care is promptly provided due to manageable numbers of TBI patients seeking care.50 The facilities are also well equipped and resourced to provide specialized care with readily available specialists. This allows maximal care, leading to improved outcomes compared to public facilities. However, high medical fees in private facilities, combined with lack of comprehensive health insurance among the poor and most vulnerable result in limited access to private facility trauma care services.19,20 Addressing gaps in capacity of public facilities has been identified as an important intervention to reduce avoidable pre-hospital mortality and morbidity burden most prevalent among the less well-off.6,10,51\n\n\nConclusion\n\nTrauma mechanism (RTI and non-RTI) and type of tertiary facility patients are transferred to (public vs private) are key factors influencing TBI mortality burden. Strengthening local trauma emergency care responses targeting RTIs and equipping public health facilities to provide quality and timely critical trauma care is indicated. Addressing post-trauma care access barriers in private tertiary facilities such as through comprehensive social insurance for the poorest and vulnerable can also significantly increase timely access to quality life-saving interventions and reduce TBI mortality burden from RTIs.\n\n\nData availability\n\nHarvard Dataverse: Association between Traumatic Brain Injury (TBI) patterns and mortality. https://doi.org/10.7910/DVN/TF4LXE52\n\nThis project contains the following underlying data:\n\n• Data Gilbert_SPSS Data-Jo- 1.tab\n\nThis project also contains the following extended data:\n\n• Medical Records Review Checklist.docx\n\nHarvard Dataverse: Replication Data for: Association between Traumatic Brain Injury (TBI) patterns and mortality. https://doi.org/10.7910/DVN/TK8BXF\n\nThis project contains the following extended data:\n\n• Data Gilbert_SPSS Data-Jo- Hav.tab\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors thanks Mr. Martin Mwenda, Ms. Winfred Kananu, Ms. Faith Ngatia and Mr. Bonface Muthomi for their assistance in data collection and reviews. We thank all health facilities and institutions which supported and participated in the study, either directly or indirectly. Special thanks to the management of KNH, Kikuyu Mission Hospital and Mater Misericordiae Hospital for institutional approval and administrative support in abstraction of patient records data in their facilities. We are greatly thankful to all persons who participated either directly or indirectly in this study.\n\n\nReferences\n\nKong SY, Shin SD, Tanaka H, et al.: Pan-Asian Trauma Outcomes Study (PATOS): Rationale and Methodology of an International and Multicenter Trauma Registry. Prehospital Emerg Care. 2017; 22(1): 1–26. PubMed Abstract \| Publisher Full Text\n\nCurtis K, McCarthy A, Mitchell R, et al.: Paediatric trauma systems and their impact on the health outcomes of severely injured children: Protocol for a mixed methods cohort study. Scand J Trauma Resusc Emerg Med. 2016; 24(1): 1–8. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdem IJ, Dare AJ, Byass P, et al.: External injuries, trauma and avoidable deaths in Agincourt, South Africa: A retrospective observational and qualitative study. BMJ Open. 2019; 9(6): 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUsselman CWNSSJRB: HHS Public Access. Physiol Behav. 2017; 176(3): 139–148.\n\nMahama MN, Kenu E, Bandoh DA, et al.: Emergency response time and pre-hospital trauma survival rate of the national ambulance service, Greater Accra (January - December 2014). BMC Emerg Med. 2018; 18(1): 3–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson MJ: A comprehensive review of the Emergency Medical Services System in Kenya.2013.\n\nThompson L, Hill M, Davies C, et al.: Identifying pre-hospital factors associated with outcome for major trauma patients in a regional trauma network: An exploratory study. Scand J Trauma Resusc Emerg Med. 2017; 25(1): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuryanto, Plummer V, Boyle M: EMS systems in lower-middle income countries: A literature review. Prehosp Disaster Med. 2017; 32(1): 64–70. PubMed Abstract \| Publisher Full Text\n\nWesson HKH, Boikhutso N, Bachani AM, et al.: The cost of injury and trauma care in low- and middle-income countries: a review of economic evidence. Health Policy Plan. 2014; 29(6): 795–808. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nTropeano MP, Spaggiari R, Ileyassoff H, et al.: A comparison of publication to TBI burden ratio of low- and middle-income countries versus high-income countries: How can we improve worldwide care of TBI? Neurosurg Focus. 2019; 47(5). PubMed Abstract \| Publisher Full Text\n\nNielsen K, Mock C, Joshipura M, et al.: Assessment of the Status of Prehospital Care in 13 Low- and Middle-Income Countries. Prehospital Emerg Care. 2013; 16(3): 381–389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrajano AD, Pereira BM, Fraga GP: Epidemiology of in-hospital trauma deaths in a Brazilian university hospital. BMC Emerg Med. 2014; 14(1): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaibo CLA, Moon TD, Joaquim OA, et al.: Analysis of trauma admission data at an urban hospital in Maputo, Mozambique. Int J Emerg Med. 2016; 9(1): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChalya PL, Mabula JB, Dass RM, et al.: Injury characteristics and outcome of road traffic crash victims at Bugando Medical Centre in Northwestern Tanzania. J Trauma Manag Outcomes. 2012; 6(1): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKinyanjui B: Traumatic Brain Injury in Kenya. SAGE Open. 2016; 6(1): 215824401663839. Reference Source\n\nOkemwa M: Patterns of injuries in road traffic accident fatalities seen at the Kenyatta National Hospital. Univ if Nairobi Res Arch. 2004; 31–2. Reference Source\n\nAdeloye D, Thompson JY, Akanbi MA, et al.: The burden of road traffic crashes, injuries and deaths in Africa: a systematic review and meta-analysis. Bull World Health Organ. 2016; 94(7). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLadeira RM, Malta DC, Morais Neto OL, et al.: Acidentes de transporte terrestre: estudo Carga Global de Doenças, Brasil e unidades federadas, 1990 e 2015. Rev Bras Epidemiol. 2017; 20(suppl 1): 157–70. Reference Source\n\nBalikuddembe JK, Ardalan A, Zavareh DK, et al.: Factors affecting the exposure, vulnerability and emergency medical service capacity for victims of road traffic incidents in Kampala Metropolitan Area: A Delphi study. BMC Emerg Med. 2017; 17(1): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoschini LP, Lu-Myers Y, Msiska N, et al.: Effect of direct and indirect transfer status on trauma mortality in sub Saharan Africa. Injury. 2016; 47(5): 1118–1122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEefect THE, Prehospital OF, Fluid I, Survival ON, Trauma OF, In P, et al: Development of a National Ems Policy for. January 2014; 2016: 4–5.\n\nMöller A, Hunter L, Kurland L, et al.: The association between hospital arrival time, transport method, prehospital time intervals, and in-hospital mortality in trauma patients presenting to Khayelitsha Hospital, Cape Town. African. J Emerg Med. 2018; 8(3): 89–94. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nKim J, Barreix M, Babcock C, et al.: Acute Care Referral Systems in Liberia: Transfer and Referral Capabilities in a Low-Income Country. Prehosp Disaster Med. 2017; 32(6): 642–650. PubMed Abstract \| Publisher Full Text\n\nStrnad M, Borovnik Lesjak V, Vujanović V, et al.: Predictors of mortality and prehospital monitoring limitations in blunt trauma patients. Biomed Res Int. 2015; 2015. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSteenkamp C, Kong VY, Clarke DL, et al.: The effect of systematic factors on the outcome of trauma laparotomy at a major trauma centre in South Africa. Ann R Coll Surg Engl. 2017; 99(7): 540–544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOno Y, Ishida T, Iwasaki Y, et al.: The off-hour effect on trauma patients requiring subspecialty intervention at a community hospital in Japan: A retrospective cohort study. Scand J Trauma Resusc Emerg Med. 2015; 23(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMock CN, Jurkovich GJ, Nii-Amon-Kotei D, et al.: Trauma mortality patterns in three nations at different economic levels: Implications for global trauma system development. J Trauma - Injury Infection and Critical Care. 1998; 44. : 804–814. PubMed Abstract \| Publisher Full Text\n\nSullivan KMS: OpenEpi - Sample Size for Unmatched Case-Control Studies.[cited 2021 Jul 9]. Reference Source\n\nLynch E: Dealing with Confounding. YouTube. 2013; Reference Source\n\nPourhoseingholi MA, Baghestani AR, Vahedi M: How to control confounding effects by statistical analysis. Gastroenterol Hepatol from Bed to Bench. 2012; 5(2): 79–83. PubMed Abstract \| Free Full Text\n\nMehmood A, Rowther AA, Kobusingye O, et al.: Assessment of pre-hospital emergency medical services in low-income settings using a health systems approach. Int J Emerg Med. 2018; 11(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nC DD: E S. Pre-hospital trauma care. Curr Opin Anaesthesiol. 2005; 14(2): 191–5. Reference Source\n\nBosson N, Redlener MA, Foltin GL, et al.: Barriers to utilization of pre-hospital emergency medical services among residents in Libreville, Gabon: A qualitative study. African J Emerg Med. 2013; 3(4): 172–177. Publisher Full Text\n\nShisoka JM: Factors that influence outcome of traumatic brain injury patients at Kenyatta National Hospital. A Dissertation Submitted in Partial Fulfillment of the Requirements for the Award of Master of Science Degree in M. 2013; (November): 1–76.\n\nBalikuddembe JK, Ardalan A, Khorasani-Zavareh D, et al.: Weaknesses and capacities affecting the Prehospital emergency care for victims of road traffic incidents in the greater Kampala metropolitan area: A cross-sectional study. BMC Emerg Med. 2017; 17(1): 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoyle RM, Harrison HL: Emergency care capacity in Freetown, Sierra Leone: A service evaluation. BMC Emerg Med. 2015; 15(1): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKobusingye OC, Hyder AA, Bishai D, et al.: Emergency medical systems in low- and middle-income countries: Recommendations for action. Bull World Health Organ. 2005; 83(8): 626–631. PubMed Abstract \| Free Full Text\n\nRithaa G: Association between Traumatic Brain Injury (TBI) patterns and mortality.2021." }	[ { "id": "94017", "date": "25 Oct 2021", "name": "Amit Agrawal", "expertise": [ "Reviewer Expertise Neurosurgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic of the current article is pertinent and is of public health importance.\nThe introduction is too long and can be shortened to include relevant details.\n\nIt will be interesting to provide the total number of cases of TBI presented to the hospital and how the unmatched sample was drawn (equal number of cases versus controls).\n\nIt will be helpful if the details of the control group can be elaborated (healthy versus others).\n\nPlease provide further details of how ICD codes were assigned or selected as it will help in future studies.\n\nPlease provide further details and numbers of excluded cases.\n\nIt will be interesting to have the details of isolated TBI versus TBI and associated injuries and their outcomes.\n\nPlease elaborate in Table 1 GCS if it is for TBI severity not for Trauma Severity; accordingly the text can be updated.\n\nIn reference to the title of Table 1: \"Patient characteristics summary by mortality outcome (cases and controls)\" - is it mortality outcome or is it a table of characteristics of the study population with subgroup distribution? Similarly, Table 2, \"Descriptive summary of trauma patterns by mortality outcomes\".\n\nIn other tables when the objective of the study is to include patients with TBI, the distribution of injuries cannot be generalized, rather it will be a distribution of associated injuries in patients with TBI. Accordingly, the language needs to be modified and sentences may need to be re-phrased.\n\nOnce these are potentially incorporated it will be great if the discussion can be modified and updated as well.\n\nPlease support the conclusion by the study findings and it should reflect the objectives of the study.\n\nFew references are incomplete and need to be updated.\n\nIt is obvious that retrospective design shall have inherent limitations, the statistical methods have text that can better be shifted to the methods section particularly “Access to life-saving…”.\n\nAlthough the study does not provide new information, however, it can be a good contribution to understanding regional (i.e. Kenya in the present case) patterns and burden of traumatic brain injury-related morbidity and mortality.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7748", "date": "31 Jan 2022", "name": "Gilbert Gilbert", "role": "Author Response", "response": "The introduction is too long and can be shortened to include relevant details We deleted literature on page 4 which presented findings on the relationship between trauma patterns and mortality outcomes and trauma statistics from high-income countries. Most of this literature was already incorporated into the discussion. This was done to make the introduction section short and concise. It will be interesting to provide the total number of cases of TBI presented to the hospital and how the unmatched sample was drawn (equal number of cases versus controls). We included the number of TBI cases and controls presented to the facilities in the study period to provide more details on the sampling frame. Please provide further details of how ICD codes were assigned or selected as it will help in future studies This was added. The codes are assigned based on the written medical notes (documentation) on patient examination and diagnosis by the clinical care providers. It will be interesting to have the details of isolated TBI versus TBI and associated injuries and their outcomes. This analytic request was not at the center of this study – is outside our scope – but details of the sampled injuries are listed in Table 3. It will be helpful if the details of the control group can be elaborated (healthy versus others) Additional details on the control group were added. Please provide further details and numbers of excluded cases. Details and the number of excluded cases have been provided. In reference to the title of Table 1: \"Patient characteristics summary by mortality outcome (cases and controls)\" - is it mortality outcome or is it a table of characteristics of the study population with subgroup distribution? Similarly, Table 2, \"Descriptive summary of trauma patterns by mortality outcomes\". In other tables when the objective of the study is to include patients with TBI, the distribution of injuries cannot be generalized, rather it will be a distribution of associated injuries in patients with TBI. Accordingly, the language needs to be modified and sentences may need to be re-phrased. The title of Table 1 was revised to indicate the data presented was on study population characteristics versus mortality distribution rather than patient characteristics and mortality outcomes. Please elaborate in Table 1 GCS if it is for TBI severity not for Trauma Severity; accordingly the text can be updated. The use of GCS scores to refer to trauma severity was replaced with TBI severity in the entire document. Once these are potentially incorporated it will be great if the discussion can be modified and updated as well. The discussion was updated with suggested details including change of trauma severity to TBI severity and use of mortality distribution instead of mortality outcomes. Please support the conclusion by the study findings and it should reflect the objectives of the study. In the conclusion, deduction on trauma patterns in which deductions on the effect of type of tertiary facility transfer on mortality outcomes was removed to retain an emphasis on association between trauma patterns and TBI mortality." } ] }, { "id": "93345", "date": "19 Jan 2022", "name": "Peter Kithuka", "expertise": [ "Reviewer Expertise Health Systems Management" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides answers to the hitherto unclear research gaps with respect to the association between traumatic brain injury patterns and mortality. This paper addresses important public health issues which have attracted limited attention in Africa over time. The authors' methodology is scientifically sound in which the findings are well anchored. The conclusions of this study will go a long way in informing important policy deliberation and further research into this area. Except for a few issues raised that need to be rephrased for clarity, the study has a robust and sound scientific base on this topic. Specifically, check on:\nBackground: The content is very relevant and logically present. However, some literature, well captured in the discussion can be cut down.\n\nStatistical Analysis: Revise the second sentence in this section to read: \"we used Pearson's chi-square test to determine the association between trauma pattern and mortality outcomes\".\nThe author can also provide more details on the control group used in this study.\n\nResults: Rephrase the first sentence in this section to read: “A descriptive summary of study population characteristics versus mortality outcomes is shown in Table 1.\"\n\nDiscussion: No comments – the discussion section is well anchored on the findings presented.\n\nConclusion: The conclusion is fairly supported by study results or data. However, the author can consider paraphrasing the first sentence to read: \"This study aimed to assess the association between trauma patterns and TBI mortality.\"\nThe conclusion should also be reviewed to focus on trauma patterns (RTI and other patterns studied) rather than the type of facility a patient is transferred to; the type of tertiary facility is presented and incorporated as a control variable in line with the statistical analysis used.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7749", "date": "31 Jan 2022", "name": "Gilbert Gilbert", "role": "Author Response", "response": "The content is very relevant and logically present. However, some literature, well captured in the discussion can be cut down. We deleted literature on page 4 which presented findings on the relationship between trauma patterns and mortality outcomes and trauma statistics from high-income countries. Most of this literature was already incorporated into the discussion. This was done to make the introduction section short and concise. The author can also provide more details on the control group used in this study. Additional details on the control group were added. Revise the second sentence in this section to read: \"we used Pearson's chi-square test to determine the association between trauma pattern and mortality outcomes\" Revision is done to read as follows, \"We used Pearson's chi-square test to determine the association between trauma pattern\". Rephrase the first sentence in this section to read: “A descriptive summary of study population characteristics versus mortality outcomes is shown in Table 1.\" The sentence was reviewed to read, \"A descriptive summary of study population characteristics and mortality distribution is shown in Table 1\". The conclusion should also be reviewed to focus on trauma patterns (RTI and other patterns studied) rather than the type of facility a patient is transferred to; the type of tertiary facility is presented and incorporated as a control variable in line with the statistical analysis used. In the conclusion, deduction on the effect of type of tertiary facility transfer on mortality outcomes was removed to retain the emphasis on the association between trauma patterns and TBI mortality." } ] } ]	1	https://f1000research.com/articles/10-795
https://f1000research.com/articles/11-122/v1	31 Jan 22	{ "type": "Software Tool Article", "title": "bit: a multipurpose collection of bioinformatics tools", "authors": [ "Michael Lee" ], "abstract": "bit is a collection of small scripts and programs that facilitate many common tasks in bioinformatics. It operates in a Unix-like command-line environment and is comprised of bash and python code. bit is openly available on GitHub, archived with Zenodo, and is conda installable. The package is useful for users who want to do things such as manipulate fasta files, calculate GC content, quickly summarize nucleotide assemblies, easily download assemblies from NCBI just based on accessions, pull amino-acid sequences from GenBank files, calculate Shannon uncertainty for columns in multiple sequence alignments, and more. The source code is hosted on GitHub: github.com/AstrobioMike/bit", "keywords": [ "bioinformatics", "toolkit", "command-line" ], "content": "Introduction\n\nThere are of course several great and widely used packages of bioinformatics helper programs already available. Some of these include the likes of seqtk,1 fastX-toolkit,2 and bbtools3 – all of which I use regularly and have facilitated goals I was trying to accomplish. But there are always more tasks that crop up that may not yet have a helper program or script already written to accomplish them. bit is a collection of small scripts and programs that were not written for any single piece of research work. Rather it is a collection that has been built (and is still being built) over several years. Anytime I need to write something to perform a task that has more than a one-off ad hoc use, something I end up using frequently, I consider adding it to the bit package. Some programs are light wrappers that extend and/or simplify the utility of existing software (like taxonkit4 and goatools5); many are written in Python leveraging the Biopython6 module (e.g. programs to summarize assemblies, calculate gc content, calculate Shannon uncertainty per column in multiple sequence alignments, pulling amino-acid sequences from GenBank files); and many are bash scripts to do things like download any assembly in different file formats from NCBI7 just by providing a list of wanted accessions. It is a rather random collection, but it is of convenience to many users.\n\n\nMethods\n\nThe package is written in Bash and Python (3+), and is built to run in a Unix-like environment.\n\nbit is packaged in conda,8 which serves as its primary means of installation. All dependencies are handled by the conda installation, but they include: python v3+; biopython6 v1.7.9+; pybedtools9 v0.8.2+; GNU parallel10 v20211022+; pandas11 v1.3.4+; entrez direct12 v16.2+; taxonkit4 v0.9.0+; goatools5 v0.8.12.\n\n\nUse cases\n\nAll commands are prefixed with ‘bit-’ and so can be seen by typing that and hitting tab twice. Each comes with a help menu by running the command with no arguments or with ‘-h’. In Figure 1 is an example with the program for downloading genome assemblies from NCBI by providing accessions.\n\n\nSoftware availability\n\nSource code available from: https://www.github.com/AstrobioMike/bit.\n\nArchived analysis code as at time of publication: https://doi.org/10.5281/zenodo.3383647.\n\nLicense: GNU GPL v3.0.", "appendix": "References\n\nLi H: seqtk.Reference Source\n\nHanon GJ: FastX-Toolkit.Reference Source\n\nBushnell B: BBtools.Reference Source\n\nShen W, Ren H: TaxonKit: a practical and efficient NCBI taxonomy toolkit.J. Genet. Genomics. 2021; 48(9): 844–850.\n\nKlopfenstein DV, Zhang L, Pedersen BS, et al.: GOATOOLS: A Python library for Gene Ontology analyses. Sci. Rep. 2018; 8: 1–17.\n\nCock PJA, Antao T, Chang JT, et al.: Biopython: Freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 2009; 25: 1422–1423. Publisher Full Text\n\nLeary NAO, Wright MW, Brister JR, et al.: Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation.2016; 44: 733–745.\n\nAnaconda-Team: Anaconda Software Distribution.2016.\n\nDale RK, Pedersen BS, Quinlan AR: Pybedtools: A flexible Python library for manipulating genomic datasets and annotations. Bioinformatics 2011; 27: 3423–3424. PubMed Abstract \| Publisher Full Text\n\nTange O: GNU Parallel.2021.\n\nTeam T pandas development: pandas. Zenodo 2020.\n\nAgarwala R, Barrett T, Beck J, et al.: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2018; 46: D8–D13." }	[ { "id": "122738", "date": "22 Feb 2022", "name": "Kai Zhang", "expertise": [ "Reviewer Expertise Bioinformatics", "single-cell genomics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author reports a collection of scripts for various bioinformatics tasks. However, the manuscript is too short and lacks important details for me to assess the significance and novelty of this work. The rationale for this study is unclear. The description of the tool is not enough. I suggest the author add more details to the Methods section and perform a thorough comparison with related tools/packages.\nSpecific comments:\nNeed to be more clear about the problems or tasks that this toolkit tries to address. How efficient are these scripts? What are the expected input sizes for your scripts? Have you done any comparison with other similar packages?\n\nIs the rationale for developing the new software tool clearly explained? No\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? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] }, { "id": "128277", "date": "23 Mar 2022", "name": "Georges Hattab", "expertise": [ "Reviewer Expertise Bioinformatics", "Artificial Intelligence", "Machine Learning", "Data Mining", "Data Visualization" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author has put some effort to compile a set of useful scripts. By looking at the number of references alone, it is clear the author lacks knowledge in the field of bioinformatics as many works are left out or not even mentioned. Aside from this fact, there is no description of the methodologies that are made available. I would invite the author to see what a normal methods section looks like. Although a practical set of scripts is provided and for the most part well written, there is a fundamental gap in describing and reproducing this work. Moreover, I would invite the author to look up relevant and openly available data sets where use cases can be presented and the usefulness can be demonstrated. In its current state, there is need for more than considerable work to improve this submission.\n\nIs the rationale for developing the new software tool clearly explained? No\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? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] } ]	1	https://f1000research.com/articles/11-122
https://f1000research.com/articles/11-121/v1	31 Jan 22	{ "type": "Review", "title": "The product science of electrically heated tobacco products: a narrative review of the scientific literature", "authors": [ "Layla Malt", "Keith Thompson", "Elizabeth Mason", "Tanvir Walele", "Thomas Nahde", "Grant O'Connell", "Keith Thompson", "Tanvir Walele", "Thomas Nahde", "Grant O'Connell" ], "abstract": "Heated tobacco products represent a novel category of tobacco products in which a tobacco consumable is heated to a temperature that releases nicotine from the tobacco leaf but not to a temperature sufficient to cause combustion. Heated tobacco products may therefore have the potential to be a less harmful alternative for adult smokers that would otherwise continue to smoke conventional cigarettes. Given the rapid development of this product category, the aim of this review was to examine the available peer-reviewed scientific evidence related to heated tobacco products and highlight any research gaps. In recent years, manufacturers of heated tobacco products have published a number of studies on their respective heated tobacco products. Whilst there is limited research that is independent of commercial interests, the available scientific evidence indicates that heated tobacco products produce a much simpler aerosol than conventional cigarette smoke, with fewer and substantially lower levels of harmful toxicants. Toxicology assessments indicate these reductions in aerosol toxicants translate to reduced biological effects. Biomarker and clinical data from studies in which product use is controlled within a clinical setting, indicate changes in biomarker levels and clinical end-points similar to observations in cessation studies, indicating the potential for reduced harm. The scientific evidence also indicates that exposure of non-users to emissions from heated tobacco products in indoor environments is significantly reduced compared to exposure resulting from smoking conventional cigarettes. Overall, the available scientific evidence indicates that heated tobacco products hold promise as a less harmful alternative to conventional cigarettes, but more independent data is required to validate industry findings. As a growing product category, epidemiological studies and independent population modelling studies are outstanding, and empirical data on how dual tobacco product category use by consumers affects their risk profile is lacking.", "keywords": [ "Heat-Not-Burn Tobacco", "Heated Tobacco Products", "Next Generation Products", "Public Health", "Risk Reduction", "Smoking", "Tobacco Harm Reduction", "Tobacco Heating Products" ], "content": "Abbreviations\n\naHTPs: Aerosol Heated Tobacco Products\n\nAIx: Augmentation Index\n\nARE: Antioxidant Response Element\n\nBAT: British American Tobacco\n\ncHTPs: Carbon Heated Tobacco Products\n\nCOPD: Chronic Obstructive Pulmonary Disease\n\nCORESTA: Cooperation Centre for Scientific Research Relative to Tobacco\n\nECG: Electrocardiogram\n\nEHCSS: Electrically Heated Cigarette Smoking System\n\neHTPs: Electrically Heated Tobacco Products\n\nETS: Environmental Tobacco Smoke\n\nEVP: e-vapour product\n\nFDA: Food and Drug Administration\n\nFEF: Forced Expiratory Flow\n\nFVC: Forced Vital Capacity\n\nHCS: High Content Screening\n\nHPHCs: Harmful and Potentially Harmful Constituents\n\nIAQ: Indoor Air Quality\n\nJTI: Japan Tobacco International\n\nKT&G: Korea Tobacco and Ginseng\n\nMOE: Margin of Exposure\n\nNGPs: Next Generation Products\n\nPMI: Philip Morris International\n\nPWV: Pulse Wave Velocity\n\nQSU: Questionnaire of Smoking Urges\n\nTHP: Tobacco Heating Product\n\nTHS: Tobacco Heating System\n\nTPM: Total Particulate Matter\n\nTSNAs: Tobacco-Specific Nitrosamines\n\n\nIntroduction\n\nThe health effects associated with conventional cigarette smoking have been extensively documented by the public health and scientific communities (United States Surgeon General, 2010; International Agency for Research on Cancer, 2012; American Cancer Society, 2018). Smoking is a cause of serious diseases in smokers, including lung cancer, heart disease and emphysema. Given recent technological advances and new innovations that allow nicotine to be decoupled from harmful tobacco smoke, tobacco product manufacturers are now developing novel product categories which may be less harmful when compared to conventional cigarettes and offer a satisfying alternative for adult smokers. For this tobacco harm reduction approach to succeed in efficiently reducing harm compared with continued conventional cigarette smoking, two criteria must be fulfilled by these non-combustible nicotine-containing next-generation products (NGPs):\n\n1. The NGP must be acceptable and satisfying for current adult smokers, so that they transition away from conventional cigarettes to the new product completely;\n\n2. The NGP must have been scientifically demonstrated to be significantly less harmful than conventional cigarettes.\n\nHowever, the potential consequences of unintended use (i.e. portions of the population for whom NGPs are not targeted towards, like youth or never smokers) must also be considered. Therefore, it is important to acknowledge that population level tobacco harm reduction can only be achieved if a scientifically-substantiated reduced harm product is accepted and used by a large number of adult smokers, who would otherwise continue to smoke, while never smokers, vulnerable populations and youth do not also begin using it. One such innovative product category within NGPs are heated tobacco products, also known as ‘heat-not-burn’ tobacco products. These products heat tobacco to a temperature that releases the nicotine and aromas from the tobacco leaf but not to a temperature high enough to cause combustion. By eliminating tobacco combustion, the levels of harmful chemicals and toxicants, including harmful and potentially harmful constituents (HPHCs), in the aerosol are substantially reduced. This in turn is expected to lead to a reduction in exposure to them for adult smokers and a subsequent reduction in toxicity in those adult smokers who use them as an alternative to conventional cigarettes. Consumer interest in heated tobacco products continues to increase as demonstrated by a rise in internet search engine searches relating to them (Caputi et al., 2017; Stall et al., 2018; Tabuchi et al., 2018) and an increased discussion of them on social media platforms (Hejlová et al., 2019; Kreitzberg et al., 2019; Jun, 2020; Barker et al., 2021).\n\nSeveral different scientific assessment programs for assessing the harm reduction potential of NGPs have been proposed (Smith et al., 2016; Levy et al., 2017; Murphy et al., 2017). Imperial Brands has developed a 5-step scientific framework as shown in Figure 1. The framework employs five distinct components and is based in part on the US National Academy of Science’s innovative blueprint for Toxicity Testing in the 21st Century which advocates for the use of in vitro testing using human cells as a more relevant alternative to traditional in vivo animal testing (Krewski et al., 2010). This narrative review will discuss the first three components of the scientific framework (product characterisation science, biological science and clinical science).\n\nHeated tobacco products developed to date have taken three distinct engineering approaches:\n\n1. Electrically-heated tobacco products (eHTPs): Use of a battery-powered handheld device which heats small non-combusted cigarette-like tobacco consumables (also known as “sticks”) which the adult smoker inserts into the device. The tobacco within the consumable is precisely heated by a number of heating elements or blades within the device to a specific temperature (Patskan and Reininghaus, 2003; Smith et al., 2016; Eaton et al., 2018).\n\n2. Aerosol heated tobacco products (aHTPs): Use of a battery-powered handheld device which heats a liquid consumable to generate a hot aerosol; this aerosol then passes through a tobacco consumable to form a tobacco-containing aerosol. These products may also be known as “hybrid” devices (Breheny et al., 2017).\n\n3. Carbon heated tobacco products (cHTPs): Use of a carbon tip which is lit by the adult smoker with a lighter or match, which heats incoming air which in turn heats a tobacco-cigarette-like product forming a tobacco flavoured aerosol containing mainly water, glycerol, nicotine and volatile tobacco components (Phillips et al., 2019).\n\nCommercially available eHTPs include ‘IQOS’, produced by Philip Morris International (PMI), ‘glo’ produced by British American Tobacco (BAT;), ‘Pulze’ produced by Imperial Brands, ‘Ploom S’ produced by Japan Tobacco International (JTI;) and ‘lil’ produced by South Korea’s KT&G (Cho and Thrasher, 2019; Lee and Lee, 2019). Further details are shown for these products in Table 1. Ploom Tech produced by JTI is an example of a commercially available aHTP, whilst there are currently no commercially available cHTPs.\n\nN/A, Not Applicable; N/S, Not Stated; THS, Tobacco Heating System; THP, Tobacco Heating Product.\n\n1 Other variants of this eHTP have been reported in the literature including THSS2.1 and THS2.4. It is assumed that these represent various generations of the handheld electrical device. THS2.2 is the most predominant designation found in the journal articles referenced in this review.\n\n2 All websites accessed on Monday 6th September 2021.\n\n3 Other Ploom products produced by JTI are regarded as being aHTPs and therefore are outside the remit of this narrative review.\n\nThe aim of this narrative review is to assess the most up to date and current available peer-reviewed scientific evidence on eHTPs and to determine if that evidence base supports their potential as a reduced harm product compared to conventional cigarettes for adult smokers that would otherwise continue to smoke. Four extensive reviews relating to heated tobacco products have already been published (Jankowski et al., 2019; Mallock et al., 2019; Simonavicius et al., 2019; Ratajczak et al., 2020) as well as a series of smaller reviews (Başaran et al., 2019; Signes-Costa et al., 2019; Znyk et al., 2021) and three journal issues published by PMI and BAT relating to their Heatbar, IQOS and glo eHTPs (Schorp et al., 2012; Smith et al., 2016; Proctor, 2018). This narrative review represents an extension of, and update to, these previous reviews.\n\nThis review will not discuss cHTPs, hybrid products (aHTPs), as such products possess engineering components of both a heated tobacco product and an e-vapour product (Glantz, 2018a) or loose-leaf tobacco vaporisers, as the design of such products permit use of non-tobacco plant-based substrates.\n\nIt should be assumed by the reader that where the descriptor “heated tobacco product(s)” is used in this review, it refers to eHTPs. It should also be noted that the descriptor used in each journal article to describe the heated tobacco product under discussion will be used in respect to that article in this review. The IQOS heated tobacco product can also described as the Tobacco Heating System (THS, THS 2.1 or THS 2.2) in the literature, the glo heated tobacco product can also described as the Tobacco Heating Product (THP1.0) whilst the Heatbar heated tobacco product (no longer commercially available) can also be described as the electrically heated cigarette smoking system (EHCSS).\n\n\nReview methodology\n\nA manual interrogation of the PubMed database was conducted on Monday 24th May 2021 to identify all potentially relevant peer-reviewed journal articles relating to eHTPs using the following six search terms: “heat-not-burn”, “heated tobacco”, “heated cigarette*”, “tobacco heating”, “heat tobacco” and “IQOS”. IQOS was used as one of the search terms as the IQOS heated tobacco product was the first modern heated tobacco product to be commercially available, is the current market leader and was expected to have a broad range of publications relating to it. For each search term used, the interrogation was limited to it being present within the title and/or abstract of the journal article with the sole additional criteria applied being that all journal articles must have been published in English. No restriction was placed on date of publication. Each of these interrogations generated a separate list of journal articles with these six lists being combined into a single journal article list to remove multiples of any journal articles identified more than once. This single combined journal article list represented the initial starting point for this narrative review.\n\nThe abstract of each journal article in this combined article list was then read by one of the authors (KT) to identify those journal articles which were irrelevant to this narrative review (those which did not discuss heated tobacco products in any capacity), those deemed to be outside the remit of this review (any journal article published in any language other than English, any journal article which discussed regulation of, policy towards or advertising of heated tobacco products or journal articles which detailed tobacco industry activities or discussed non-peer reviewed internal tobacco industry documents), those which related to heated tobacco products which utilise a carbon heat source (cHTPs), those which related to hybrid products (also known as aHTPs and/or loose-leaf tobacco vaporisers and those which discussed the behavioural science of heated tobacco product use (which will be covered in a separate review article).\n\nAdditional relevant peer-reviewed journal articles were identified through manual review, analysis and cross-linking of reference lists contained in journal articles identified as described above. Conference posters, conference proceedings and journal abstracts (reported in the absence of an associated journal article) were excluded due to their lack of peer-review.\n\nThe identified relevant articles were reviewed in full by one of the authors (KT) to determine the key themes relevant to this review and extract the relevant data. Some articles contained data relevant to more than one end-point and so are discussed in more than one section.\n\nAutomatic e-mail alerts were created within the PubMed database for the search terms indicated above on Monday 24th May 2021 to identify relevant journal articles published between that date and Tuesday 31st August 2021 (with this latter date representing the cut-off date for inclusion in this narrative review). These additional journal articles were then included in the final list of journal articles referenced in the narrative review.\n\nCitation details for all journal articles identified as discussed above are shown in the Underlying data (Supplementary File 1) (Mason, 2021). The overall search strategy and the numbers of journal articles identified at each stage of the search process are shown in Figure 2.\n\n1The search strategy identified two separate citations for the Ioakeimidis et al. (2020) article, one of which was subsequently removed from the PubMed database. As such, the refined article list above is shown to contain 255 articles rather than 256 articles. See Underlying data: Supplementary File 1 for further details.\n\n\nAerosol characterisation\n\nThis section of the review will discuss those articles that have reported on the characterisation of aerosols experimentally produced from heated tobacco products, both in terms of their physical properties and their chemical constituents.\n\nCharacterisation of the aerosol produced by the THS 2.2 heated tobacco product and mainstream smoke produced by the 3R4F Kentucky Reference cigarette with respect to particle size indicated that the mean mass median aerodynamic diameters were 0.7μm and 0.8μm respectively based on ten replicate samples for each (Schaller et al., 2016a). In every instance, the upper boundary for particle size were below 2.5μm and as a result it was estimated that both the aerosol from the heated tobacco product and mainstream smoke from the 3R4F Kentucky Reference cigarette were respirable with a margin of error of 5%; more than 85% of the aerosol droplets could be reasonably expected to reach the alveoli of the lung. A subsequent study characterised the particulates released by the IQOS heated tobacco device combined with four different stick variants (Pacitto et al., 2018). Median particle number concentration ranged from 7.04×107 to 9.64×107 particles/cm3, surface area concentrations ranged from 2.04×1012 to 5.08×1012 nm2/cm3 whilst the mode of the particle number distribution ranged from 93 to 108nm. No statistically significant differences were observed between the different stick variants. Characterisation of the aerosol produced by the non-mentholated variant of the THP 1.0 heated tobacco product and mainstream smoke produced by the 3R4F Kentucky Reference cigarette with respect to particle size indicated mass median diameters of 329±50nm and 272±19nm respectively with geometric standard deviations of 1.80±0.06 and 1.42±0.03, respectively (Forster et al., 2018a). Count median diameters for the non-mentholated variant of the heated tobacco product and mainstream smoke produced by the 3R4F Kentucky Reference cigarette were 39±9nm and 186±12nm respectively, with total particulate numbers per puff of 5.26×1010±1.77×1010 and 3.6×1011±5.9×1010, respectively (Forster et al., 2018a).\n\nAnalysis of the aerosol produced by the THS 2.2 heated tobacco product and the mainstream smoke produced by the 3R4F Kentucky Reference cigarette indicated that whilst the conventional cigarette released significant quantities of solid carbon particles (1012 particles present in mainstream smoke over eleven puffs), no such particles were detected with the heated tobacco product, which was suggested by the authors to indicate the absence of both combustion and pyrolysis (Pratte et al., 2017). A subsequent study published by the same authors further corroborated this finding using a methodology involving a thermo-denuder operating at 300°C (Pratte et al., 2018). Results from this study indicated that whilst mainstream smoke from the 3R4F Kentucky Reference cigarettes contained solid particles or high boiling point droplets far above the lower limit of quantification of the analytical method employed, the aerosol produced from the THS 2.2 heated tobacco product did not. The absence of combustion in such products is corroborated by observations of no meaningful increase in levels of exhaled carbon monoxide in adult smoker volunteers after use of several different heated tobacco products under controlled laboratory conditions (Adriaens et al., 2018; Caponnetto et al., 2018; Maloney et al., 2021) and statistically significant decreases in exhaled carbon monoxide levels in adult smokers, not wishing to quit, who switched to a heated tobacco product for a period of six months (Beatrice and Massaro, 2019).\n\nThis section of the review will detail those studies which have attempted to quantify the chemical constituents present in the aerosol of heated tobacco products when operated under experimentally controlled laboratory conditions. In the majority of studies discussed, the Health Canada Intense (ISO intense) analytical machine puffing regime has been widely used with some modifications given the absence of testing standards specific for heated tobacco products (Belushkin et al., 2018). It is apparent that detailed “real-world” puffing topography data is required in order to identify the most appropriate puffing parameters for heated tobacco products (McAdam et al., 2019).\n\nThis section of the review will not discuss those studies which have characterised flavour ingredients present in heated tobacco products (including menthol) (Reger et al., 2018; Jaccard et al., 2019a) or investigated their transfer rates to aerosol (Czégény et al., 2016; Blazsó et al., 2018). Toxicological studies have suggested that the presence of flavour ingredients does not modify the in vitro effects of heated tobacco product aerosols (Crooks et al., 2018; Le Godec et al., 2019). Nevertheless, separate authors have indicated a need for additional research on flavour ingredients in heated tobacco products (Kaur et al., 2018). This section of the review will, however, separately discuss nicotine and tobacco-specific nitrosamines (TSNAs) as articles specifically quantifying these chemical constituents have been published.\n\nUsing experimental designs where tobacco samples have been heated under precisely controlled conditions, it has been shown that yields of several chemical constituents are markedly reduced at lower temperatures (Torikai et al., 2004; Zhou et al., 2015; Forster et al., 2015). Using a sample of commercial blended tobacco, the yields of hydrogen cyanide, benzo [a] pyrene, formaldehyde, acrolein, isoprene, styrene, phenol and 1-aminonaphthalene were quantified as the sample was heated to either 300°C, 500°C, 800°C or 1000°C either in an air or nitrogen atmosphere (Torikai et al., 2004). In the case of each smoke constituent, yields increased significantly as the temperature increased in both atmospheres. When a range of samples of flue-cured tobacco where heated in a furnace with constant sample weight and air velocity and the temperature varied between 350°C and 750°C, it was observed that carbon monoxide yield was remarkably low when compared to yields seen with lit or smouldering tobacco (Zhou et al., 2015). A subsequent study employed a bench-top furnace to heat tobacco samples to between 100 and 200°C in 20°C increments and quantified the yields of numerous HPHCs released by the tobacco (Forster et al., 2015). Water, total and nicotine-free dry particulate matter and three TSNAs (NNN, NNK and NAT) were found at all temperatures tested. Several HPHCs were not detected at the lowest temperatures but were found as the temperature increased incrementally up to 200°C (nicotine, carbon monoxide, acetaldehyde, crotonaldehyde, formaldehyde, NAB, acetone, butyraldehyde, methyl ethyl ketone and propionaldehyde) whilst others were not detected at any temperature tested including ammonia, acrolein, hydrogen cyanide, phenol, benzene, 1,3-butadiene, isoprene and toluene.\n\nThe most comprehensive study currently available on the chemical characterisation of experimentally produced aerosols from heated tobacco products identified a total of 529 chemical constituents (excluding water, glycerol and nicotine) in the aerosol of the IQOS heated tobacco product at concentrations exceeding a concentration 100ng per stick (Bentley et al., 2020). The majority of the chemical constituents (n=402) were identified as being present in the particulate phase of the aerosol with thirty-nine of these being present in both the particulate and gas/vapour phases. The identification of 80% of the chemical constituents (representing more than 96% of the total determined mass) was confirmed using a range of analytical methodologies. Separate authors identified a total of 205 chemical constituents in the particulate phase of the aerosol derived from the same heated tobacco product (Savareear et al., 2017). Seventeen different chemical classes were identified including ketones (n=34), alcohols (n=31), aldehydes (n=22) and alicyclic hydrocarbons (n=20).\n\nUsing a similar approach on the vapour phase of the THP1.0/glo heated tobacco product, the same research group identified a total of 85 and 202 chemical constituents in the aerosol from the THP1.0/glo heated tobacco product and 3R4F Kentucky Reference cigarette respectively (Savareear et al., 2018). Thirty-five chemical constituents were found to be common to the vapour phase of both products. With respect to quantification of individual chemical constituents, a subsequent study analysed 126 chemical constituents in the aerosol from the THP1.0 heated tobacco product and directly compared these results with the 3R4F Kentucky Reference cigarette (Forster et al., 2018a). For the 102 chemical constituents which were detected in the aerosol of the THP1.0 heated tobacco product and in the mainstream smoke of the 3R4F Kentucky Reference cigarette, an overall average reduction of >95% was observed with the heated tobacco product. Data for the other 24 chemical constituents were excluded from the reduction calculations as their concentrations were below the limit of quantification in the aerosol from the heated tobacco product or mainstream smoke from the 3R4F Kentucky Reference cigarette or both. With respect to the particulate phase (and specifically the volatile organic compounds) of the aerosol produced from the THP1.0 heated tobacco product compared to mainstream smoke of the 3R4F Kentucky Reference cigarette, a total of 160 and 592 peaks were identified respectively using a methodology combining two-dimensional gas chromatography and time of flight mass spectrometry detection (Savareear et al., 2019). Ninety-three compounds were common to both sample preparations and in the vast majority of cases, observed levels were significantly lower with the heated tobacco product than with the reference cigarette. Aside from glycerine and its monoacetate, only nine compounds had marginally higher concentrations in the heated tobacco product aerosol when compared to mainstream smoke from the 3R4F Kentucky Reference cigarette with the increase being ≤1μg between cigarette and stick (methylene chloride, hexane, 2-methylfuran, 1H-pyrrole, 2,5-furandione, 2-furanmethanol, 4-cyclopentene-1,3-dione, dihydro-2(3H)-furanone and alpha-monopropionin).\n\nIn 2012, the United States Food and Drug Administration (FDA) published a list of 93 HPHCs present in tobacco products and tobacco smoke (US FDA, 2012). Table 2 shows data relating to these HPHCs with respect to their experimentally determined levels in the aerosol of heated tobacco products with direct comparison to their levels in the mainstream smoke of conventional cigarettes. Comparative data between heated tobacco products and conventional cigarettes is either based on direct quantitative results for both product groups reported within the same article, quantitative data reported elsewhere within the scientific literature for the conventional cigarette comparison, or a combination of both. Other approaches quantified HPHCs within aerosols experimentally produced from heated tobacco products without discussion of conventional cigarettes; in such cases, data can be extracted where the HPHC was determined to be below either the limit of detection or limit of quantification of the analytical method employed (Poget et al., 2017).\n\nReferences on which this table is formulated are shown in Supplementary File 2 (Underlying data (Mason, 2021). AD, Addictive; CA, Carcinogen; CT, Cardiovascular Toxicant; Glu-P-1, 2-amino-6-methyldipyrido[1,2-a:3’,2’-d]imidazole; Glu-P-2, 2-aminodipyrido[1,2-a:3’,2’-d]imidazole; HPHC, Hazardous and Potentially Hazardous Constituent; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; LOD, Limit of Detection; LOQ, Limit of Quantification; MeA-α-C, 2-amino-3-methyl)-9H-pyrido[2,3-b]indole; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PhlP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; RDT, Reproductive or Developmental Toxicant; RT, Respiratory Toxicant; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 1-methyl-3-amino-5H-pyrido[4,3-b]indole.\n\nAs indicated in Table 2, findings reported in the scientific literature with respect to HPHCs are categorised into five groups. Three of the groups are based on whether the individual HPHC has been experimentally quantified at levels lower than, comparable to or higher than those observed with conventional cigarette smoke. The remaining two groups are for those results where the experimentally determined value for the HPHC was reported as being below either the limit of detection and/or limit of quantification of the analytical method reported in the original article and those HPHCs for which no experimentally determined data could be identified. Table 2 shows that, for the 80 out of 86 (93%) of those HPHCs having data reported in the literature, levels observed with heated tobacco product aerosols are either below the limit of detection and/or limit of quantification of the analytical method employed, or substantially lower than those levels observed with conventional cigarette smoke. These observations have been reported to be maintained across a range of various high intensity puffing regimes for the THS 2.2 heated tobacco product based on a subset of 54 HPHCs with observed reductions being consistently more than 90% (Goujon et al., 2020). In addition, these quantitative results agree with separate data published within the scientific literature solely in a qualitative fashion where analysis of the IQOS heated tobacco product and 3R4F Kentucky reference cigarette GC-MS chromatographic fingerprints indicated a non-nicotine global component reduction of greater than 80% for the IQOS heated tobacco product in comparison to the 3R4F Kentucky reference cigarette (Ibañez et al., 2019). For three of the HPHCs (aflatoxin B1, coumarin and N-nitrososarcosine) no standardised methods have been developed to date for the quantification of these HPHCs in either conventional cigarette smoke or aerosols from heated tobacco products, and as such, no data is reported in Table 2 (Forster et al., 2018a). The single experimental value for N-nitrosodiethanolamine suggesting higher levels with a heated tobacco product than with a conventional cigarette was reported by the original authors as being due to background contamination (Forster et al., 2018a). No data has been reported for the three radioactive HPHCs (polonium-210, uranium-235 and uranium-238) or for chlorinated dioxins/furans (although data has been reported for furan) (Crooks et al., 2018; Forster et al., 2018a). With respect to those results for individual HPHCs where specific studies have found higher levels with a heated tobacco product than with conventional cigarettes, and with this being in opposition to the other results available in the scientific literature, these results are associated with historical heated tobacco products which are no longer commercially available. Two specific exceptions are for chromium (Forster et al., 2018a) and benzo [c] phenanthrene (Dusautoir et al., 2021) where the reported results were obtained using the THP1.0 and IQOS heated tobacco products respectively.\n\nThe significant majority of studies reported in Table 2 used the 3R4F Kentucky reference cigarette as their comparator cigarette. The 3R4F Kentucky reference cigarette serves as an international standard for research purposes and was approved by representatives of commercial manufacturers. The 3R4F Kentucky reference is not commercially available and not intended for use by ‘real-world’ adult smokers. Furthermore, the 3R4F Kentucky reference cigarette was produced at a single time point as a single batch using a single set of tobacco blends. In light of these potential limitations, separate authors have compared the yields of HPHCs in the THS 2.2 heated tobacco product with the 3R4F Kentucky refence cigarette as well as an extensive range of commercial conventional cigarettes from numerous countries purchased in different years (Jaccard et al., 2017). The results from this study, where comparison was made with commercially available products, were found to be highly comparable to the reductions seen in HPHC levels (approximately 90%) when compared to the 3R4F Kentucky reference cigarette. Changes to the blend composition used in the tobacco of the THS 2.2 heated tobacco product has been demonstrated to have a minimal effect on the levels of HPHCs in the aerosol subsequently produced by the product (Schaller et al., 2016b) whilst comparison between two tobacco blends (Burley and Virginia) have indicated comparable constituents being released after heating of the tobacco (Davies et al., 2018). It should also be noted that stocks of the 3R4F Kentucky reference cigarette are almost depleted. A new Kentucky reference cigarette, the 1R6F, has been produced and early initial data suggests comparable results for reductions in HPHCs for the THS 2.2 heated tobacco product compared to the 3R4F Kentucky reference cigarette (Jaccard et al., 2019b).\n\nWith respect to chemical constituents present in the aerosol of heated tobacco products which are not listed on the 2012 US FDA HPHC list, the significant majority of studies have indicated that levels of numerous chemical constituents such as free radicals (Shein and Jeschke, 2019; Bitzer et al., 2020) are present at lower concentrations in the aerosol of heated tobacco products compared to concentrations found in conventional cigarette mainstream smoke. Nevertheless, several chemical constituents have been reported to be present in higher concentrations in the aerosol of heated tobacco products compared to mainstream smoke from conventional cigarettes under machine-generated experimental conditions (including valeraldehyde, glyoxal, methyl glyoxal and acenaphthene). Valeraldehyde, glyoxal, methyl glyoxal were reported to be absent from the mainstream smoke from Marlboro Red conventional cigarettes whilst present in quantifiable levels in the aerosol from the IQOS heated tobacco product (Salman et al., 2019) whilst a second study reported all three chemical constituents to be present in aerosols from two heated tobacco products (IQOS and glo) and three reference cigarettes [3R4F, 1R5F and CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco) CM6 test-piece] with levels typically lower with heated tobacco products (Uchiyama et al., 2018). The observation of valeraldehyde, glyoxal, methyl glyoxal at higher levels in aerosol from the heated tobacco product than in the mainstream smoke from the Marlboro Red cigarette can likely be explained. Valeraldehyde is present due to the thermal degradation of flavour compounds present in the tobacco rod of the heated tobacco product which are not present in the tobacco rod of the conventional cigarette and glyoxal and methylglyoxal are present due to the thermal degradation of the propylene glycol present in the tobacco rod of the heated tobacco product. Acenaphthene was reported to be present in higher concentrations in the aerosol from a heated tobacco product compared to mainstream smoke from conventional cigarettes, albeit when assessed using a non-standardised testing methodology (Auer et al., 2017a). Separate researchers have indicated that the observed levels of acenaphthene, in both conventional cigarettes and heated tobacco products, do not pose a meaningful toxicological risk to smokers (Lachenmeier et al., 2018). Significant limitations of the experimental methodologies and analytical methods used to quantify the reported levels by Auer et al. have been subsequently highlighted by separate researchers (Caruso and Polosa, 2017; Maeder and Peitsch, 2017) but defended by the original researchers (Auer et al., 2017b).\n\nNicotine levels present in heated tobacco products have been reported to be largely comparable to those observed with conventional cigarettes (Jaccard et al., 2017). Numerous studies have indicated that levels of nicotine in both tobacco filler (Bekki et al., 2017) and aerosol (Auer et al., 2017a; Bekki et al., 2017; Farsalinos et al., 2018a; Salman et al., 2019) are highly similar to those with conventional cigarettes confirming this aspect of the product design.\n\nNicotine-related alkaloids (nornicotine, anatabine, anabasine and myosmine) have been demonstrated to be present in both the tobacco filler and aerosol produced from three different heated tobacco products, none of which were identified by their commercial names (Jeong et al., 2018). Cotinine, whilst detected in the tobacco filler of the 3R4F reference cigarette and CORESTA Monitor (CM7) test piece, was not detected in either the tobacco filler or aerosol produced from the heated tobacco products (the presence or otherwise of cotinine in the mainstream smoke of the two reference cigarettes was not determined) (Jeong et al., 2018).\n\nThe presence of free-base nicotine within the aerosol produced by heated tobacco products is uncertain at this time. One study indicated that levels of free-base nicotine within the aerosol of the IQOS heated tobacco product were comparable to those found in conventional cigarette mainstream smoke when analysed under two different aerosol-generation regimes (Salman et al., 2019), whilst a second study failed to detect any meaningful levels of free-base nicotine in the aerosol from several variants of the same heated tobacco product (Meehan-Atrash et al., 2019). The pH of aerosol produced by the IQOS heated tobacco product and mainstream smoke produced from conventional cigarettes were found to be largely comparable (Salman et al., 2019) suggesting that a meaningful difference in the relative percentages of the various protonated forms of nicotine between the heated tobacco product and conventional cigarettes is unlikely. There is some evidence from a theoretical mechanistic study to suggest that nicotine degradation may be more rapidly initiated as temperature is reduced which was proposed by the authors to suggest that heated tobacco products may be more likely to experience nicotine degradation than conventional cigarettes (Chavarrio Cañas et al., 2021).\n\nSeveral articles have identified the presence of four TSNAs (NNN, NAT, NAB and NNK) both in the tobacco filler (Bekki et al., 2017; Jaccard et al., 2018; Jeong et al., 2018) and in the aerosols generated from heated tobacco products [only NNN and NNK are included in the 2012 US FDA HPHC list] (Bekki et al., 2017; Jaccard et al., 2018; Jeong et al., 2018; Leigh et al., 2018a; Ishizaki and Kataoka, 2019; Li et al., 2019). When compared on the basis of the sum of the four main TSNAs investigated (NNN, NAT, NAB and NNK), levels observed in the aerosols of the IQOS heated tobacco product were significantly lower than those observed in conventional cigarette smoke (Bekki et al., 2017; Li et al., 2019).\n\nWith respect to chemical constituents and specifically HPHCs, the data shown in Table 2 indicates that experimentally produced aerosols from heated tobacco products contain fewer and significantly lower levels of these chemical constituents compared to conventional cigarette mainstream smoke in almost all instances. Whilst some non-HPHC chemical constituents have been identified in aerosols from heated tobacco products at levels higher than those observed with conventional cigarette mainstream smoke, or have been quantified in aerosols from heated tobacco products but not in conventional cigarette mainstream smoke at all, the significance of these analytical observations to the toxicological profile of heated tobacco products remains unknown and requires further investigation (St Helen et al., 2018). These conclusions are in agreement with those of a separate review on the chemical content of aerosols from heated tobacco products which concluded that “the concentrations of chemical compounds in the aerosol were about 10 times lower than that of cigarette smoke” and that “replacing smoking in the traditional way by heating tobacco modified significantly the content of chemical substances found in aerosol” (Szparaga et al., 2021).\n\nWith respect to the physical characterisation of aerosols produced from heated tobacco products, it is evident that such aerosols differ significantly from conventional cigarette mainstream smoke. This conclusion is an agreement with those of a separate experimental study which investigated the suitability of existing analytical laboratory processes for use with heated tobacco products (Gasparyan et al., 2018). The authors observed significant differences for each characterising variable between the heated tobacco products under investigation (THS 2.2 and THP 1.0) and those values typically observed with both conventional cigarettes and e-vapour products (EVPs) suggesting that the aerosol from the heated tobacco products was fundamentally distinct in terms of its physical properties. The authors concluded that “taken collectively with other available aerosol chemistry and biological results on heated tobacco products in the literature, they show a fundamentally different aerosol in heated tobacco products and call for category-specific product standards and terminology” (Gasparyan et al., 2018).\n\n\nIn vivo toxicology\n\nThis section of the review will discuss articles which have reported in vivo toxicological and histopathological effects of heated tobacco products. With the exception of a single mouse skin painting study, all of the available studies have exposed rodents to experimentally generated aerosols from heated tobacco products via inhalation using either a nose-only or whole-body experimental regime. It is important to note that the relevance of in vivo studies for assessing the effects of heated tobacco product aerosols for human exposure is unclear and results should be interpreted within this context.\n\nThe sole mouse skin painting study (conducted as part of a larger toxicological investigation) compared the carcinogenic potential of total particulate matter from the EHCSS Series K heated tobacco product (also known as Heatbar) with total particulate matter produced from the 2R4F Kentucky reference cigarette and two commercially available conventional cigarettes (Marlboro Lights and Marlboro Ultra Lights) over a 26-week exposure period using three dose levels (30mg, 60mg or 90mg total particulate matter per week) (Werley et al., 2008). No tumours were observed in the control group treated solely with dimethylbenzanthracene (the vehicle used for administration of the condensates). Condensate produced from the heated tobacco product demonstrated later dermal tumor onset, lower dermal tumor incidence, reduced dermal tumor multiplicity and a lower proportion of malignant dermal tumors than condensates produced from the conventional cigarettes. Treatment with a weekly dose of 30mg total particulate matter per week from the heated tobacco product showed a mean value for tumour multiplicity (number of tumours per mouse at the end of the application period), which was approximately 24% of the group given an equivalent dose from the 2R4F Kentucky reference cigarette. Non-statistically significant decreases were seen for the remaining two dose levels.\n\nTo date, there have only been two in vivo studies reported that have utilised an acute exposure experimental methodology involving a heated tobacco product.\n\nThe first study investigated the effect of acute exposure to mainstream smoke from a commercial cigarette (Marlboro Red) or aerosol from a heated tobacco product (IQOS) on vascular endothelial function using flow-mediated dilation as an experimental surrogate (Nabavizadeh et al., 2018). Anaesthetised male Sprague-Dawley rats (n=8 per group) were exposed via a nose cone to mainstream smoke from the Marlboro Red cigarette, aerosol from the IQOS heated tobacco product or fresh air (as a control group). The exposure regime consisted of a series of consecutive thirty-second cycles, each consisting of either five or fifteen seconds of exposure followed by removal of the nose cone for the remainder of the thirty second cycle. Each rat was exposed to either ten cycles over five minutes or three cycles over ninety seconds to approximate exposure to a single heated tobacco product use. Flow-mediated dilation (as an experimental estimate of vascular endothelial function) in the femoral artery was measured before and after exposure after temporary surgical occlusion of the common iliac artery. Flow-mediated dilation was reduced comparably by ten fifteen-second exposures to heated tobacco product aerosol and conventional cigarette smoke but not by exposure to fresh air. Flow-mediated dilation was also reduced comparably by ten five-second exposures to heated tobacco product aerosol and conventional cigarette smoke but not by exposure to fresh air. No statistical analyses were conducted to determine whether or not differences in the observed effects between the heated tobacco product and the conventional cigarette were statistically significant or not.\n\nThe second study investigated the effects of prenatal exposure to experimentally generated aerosol from the IQOS heated tobacco product, mainstream smoke from the 3R4F Kentucky reference cigarette or filtered air on testicular function in male offspring (Yoshida et al., 2020). Pregnant CD-1 mice (n=10 per group) were exposed using a whole-body exposure system for two 20-minute periods on days seven and fourteen of gestation. Adult male offspring were then divided into six groups based on the respective exposure history of the mothers and their age (five and fifteen-weeks old; age-points at which the male mice were sexually immature and sexually mature respectively). Spermatogenesis, sperm characteristics, serum testosterone and seminiferous morphology were evaluated. Prenatal exposure to experimentally produced aerosol from the IQOS heated tobacco product increased abnormal seminiferous tubule morphology and decreased sperm production at five week of age, but not at fifteen weeks of age, whilst exposure to mainstream smoke from the 3R4F Kentucky Reference cigarette did not. There were no statistically significant differences between the two exposed groups with regard to fertility, gestation period length, litter size or sex ratio at birth. There were no statistically significant differences in body weight, testicular weight, epididymis weight or serum sex hormones levels of the male offspring when stratified on the basis of the exposure group of their mothers.\n\nNumerous chronic rodent exposure studies investigating the biological and histopathological effects of exposure to experimentally produced aerosols from the EHCSSS and THS 2.1/THS2.2 heated tobacco products (also known as Heatbar and IQOS respectively) have been published by PMI. In addition to those chronic rodent exposure studies reported by PMI, four studies undertaken by independent researchers have been published within the last year (Bhat et al., 2021; Scharf et al., 2021; Vivarelli et al., 2021; Daou et al., 2021) as well as the independent development of an aerosol exposure apparatus for in vivo experiments using heated tobacco products (Sawa et al., 2021).\n\nThe studies published by PMI have combined classical histopathological end-points with a Systems Toxicology Assessment to assess the transcriptomic and proteomic effects of exposure to experimentally generated aerosols from the various heated tobacco products on specific tissues, organs and the expression of specific proteins and/or nucleic acid species, primarily in the Apo E-/- mouse. In every instance, the reported effects have been directly compared to those observed using the 3R4F Kentucky reference cigarette as a comparison product. Exposure to aerosols has been on comparable nicotine concentrations (expressed either as mg/m3 or μg/L) with several concentrations used for either or both of the aerosols. Exposure periods have ranged from thirty-five and/or ninety days (Terpstra et al., 2003; Moennikes et al., 2008; Werley et al., 2008; Oviedo et al., 2016; Wong et al., 2016) to six months or more [not including any additional recovery periods] (Phillips et al., 2016, 2019; Lo Sasso et al., 2016a; Titz et al., 2016, 2020a; Szostak et al., 2017; Choukrallah et al., 2019; Szostak et al., 2020; Wong et al., 2020; Battey et al., 2021). The suitability of the Apo E-/- mouse as an experimental model for studying the effects of both conventional cigarette smoke and heated tobacco product aerosol exposures on cardiovascular and respiratory disease end-points has been discussed elsewhere by separate authors (Lo Sasso et al., 2016b).\n\nWith respect to the findings of these studies, classical histopathological analyses have indicated that exposure to aerosol from heated tobacco products consistently yielded significantly reduced biological effects when compared to those observed with conventional cigarettes in a range of tissues including the respiratory tract (Phillips et al., 2016, 2019; Titz et al., 2016, 2020a; Oviedo et al., 2016; Wong et al., 2016), cardiovascular system (Phillips et al., 2016; 2019; Szostak et al., 2020), liver (Lo Sasso et al., 2016a; Wong et al., 2016) and gastrointestinal tract (Battey et al., 2021). Observations from a life-time exposure study using A/J mice with an 18-month exposure period indicated that chronic exposure to aerosol from the THS 2.2 heated tobacco product did not increase the incidence or multiplicity of bronchioalveolar adenomas or carcinomas relative to sham-exposed mice whilst exposure to mainstream smoke from the 3R4F Kentucky reference cigarette did (Wong et al., 2020). The findings of one inhalation study (Wong et al., 2016) have been re-interpreted by separate authors to suggest that exposure to aerosol from the THS 2.2 heated tobacco product, but not to mainstream smoke from the 3R4F Kentucky reference cigarette, may have resulted in hepatotoxicity in the exposed rats based on a re-analysis of alanine aminotransferase enzymatic activity levels, liver weights and hepatocellular vacuolisation as reported in the original article (Chun et al., 2018). It should be noted, however, that a previous study (Lo Sasso et al., 2016a), which primarily focused on the potential effects of aerosol exposure from the same heated tobacco product on hepatic tissue, did not demonstrate any gross indicators for hepatotoxicity in its results.\n\nResults from the Systems Toxicology Assessment of tissues obtained from the majority of the studies conducted by PMI have indicated a significantly lower response in terms of gene, microRNA and/or protein expression as a result of exposure to experimentally generated aerosols from heated tobacco products compared to exposure to mainstream smoke from the 3R4F Kentucky Reference cigarette (Kogel et al., 2016; Elamin et al., 2016; Lo Sasso et al., 2016a; Sewer et al., 2016; Szostak et al., 2017, 2020; Choukrallah et al., 2019; 2020; Lavrynenko et al., 2020; Titz et al., 2020b; van der Plas et al., 2020).\n\nWith respect to those studies published by independent researchers, three concluded that exposure to aerosols produced from the IQOS heated tobacco product produced detrimental effects on the respiratory system which were comparable, in some instances, to those effects seen with conventional cigarette mainstream smoke (Bhat et al., 2021; Vivarelli et al., 2021; Daou et al., 2021) whilst the fourth reported significantly higher levels of metallothionein I and II expression after exposure to conventional cigarette mainstream smoke than after exposure to aerosol from the IQOS heated tobacco product (Scharf et al., 2021). In the first study, male and female C57BL/6NCr mice were exposed to experimentally-generated aerosol from the IQOS heated tobacco product or mainstream smoke from the 3R4F Kentucky Reference cigarette for a total of 5 hours per day for 2 weeks with 20 sticks being consumed each day (Bhat et al., 2021). Mice were exposed in a whole-body exposure system with aerosol being generated using the Health Canada Intense smoking regime. Exposure to aerosol from the IQOS heated tobacco product and mainstream smoke from the 3R4F Kentucky reference cigarette was reported to result in significantly increased levels of albumin in bronchoalveolar lavage fluid compared with air-exposed controls (reported to be a surrogate marker for lung epithelial cell damage). Albumin levels were lower after exposure to aerosol from the IQOS heated tobacco product than after exposure to mainstream smoke from the 3R4F Kentucky reference cigarette, although still significantly higher than those observed with controls, indicating lower lung damage with the IQOS heated tobacco product. Both exposure scenarios also produced increased infiltration of immune cells to the lungs of the mice compared with controls. Levels of neutrophils after exposure to IQOS aerosol showed no statistically significant differences when compared to controls whilst levels of macrophages were significantly decreased. In the second study, Sprague-Dawley rats were whole-body exposed to aerosol from the IQOS heated tobacco product for four weeks (Vivarelli et al., 2021). Rats were exposed to the aerosol produced from eight sticks per day, for five consecutive days per week for four weeks. Analysis of tracheal tissue using scanning electron microscopy showed marked changes from those observed in fresh air controls including the presence of erythrocytes and necrotic cells on the epithelial surface. Exposure was also indicated to increase expression of IL-13, IL-10, IL-12, TNF-α and INF-δ. Urinary mutagenicity was also significantly increased after exposure to IQOS aerosol compared to controls when assessed using the Ames test in the presence of S9 mixture. This study did not include a comparable treatment group of rats exposed to conventional cigarette mainstream smoke. In the third study, diabetic and non-diabetic mice were exposed to aerosol from the IQOS heated tobacco product or mainstream smoke from the 3R4F Kentucky reference cigarette using a nose-only regime (Daou et al., 2021). Mice were exposed for seven consecutive days with two three-hour exposure sessions per day. Exposure to aerosol from the heated tobacco product did not produce any significant oxidative stress or increase in apoptosis in either diabetic or non-diabetic mice whereas exposure to conventional cigarette mainstream smoke did. Exposure to heated tobacco product aerosol did produce an increase in albumin levels in bronchoalveolar lavage fluid in both diabetic and non-diabetic mice and an increase in TNF-α and IL-1β levels in diabetic mice only. In the fourth study, C57BL/6 mice were exposed to filtered air (as a control), to mainstream smoke from Marlboro Red conventional cigarettes or aerosol from the IQOS heated tobacco product (Scharf et al., 2021). Both products were smoked using the Health Canada Intense regime and mice were exposed for one hour twice per day for five days prior to sacrifice. The number of conventional cigarettes and sticks used was matched by the amount of nicotine for each hour of exposure. Analysis of liver and lung tissue collected sixteen hours after the final exposure indicated that metallothionein I and II expression was markedly enhanced in lung and liver tissue collected from mice exposed to conventional cigarette mainstream smoke than in tissues collected from mice exposed to filtered air or aerosol from the IQOS heated tobacco product with no statistically significant difference between these two groups.\n\nThere appears to be a significant disparity in regard to the reported findings of in vivo studies investigating heated tobacco products. Those studies published by heated tobacco product manufacturers have indicated that exposure to aerosol from heated tobacco products consistently yielded significantly reduced biological effects when compared to those observed with conventional cigarettes in a range of tissues including the respiratory tract based on both histopathological and toxicological analyses. In opposition, the small number of studies published by independent researchers have concluded that exposure to aerosol from heated tobacco products produced comparable effects to those seen with conventional cigarettes. This disparity may be due to the use of different species between studies (PMI typically used the A/J or Apo E-/- mouse in their studies compared to the Sprague-Dawley rat, CD-1 mouse or C57BL/6NCr mouse used in the independent studies) and/or use of different exposure methodologies. Specifically, it should be noted that whilst PMI investigated the effects of conventional cigarette mainstream smoke and heated tobacco product aerosol using comparable nicotine concentrations, the independent studies did not and instead compare the effects using exposure to mainstream smoke or aerosol produced from equivalent numbers of cigarettes/sticks per exposure session (Yoshida et al., 2020; Bhat et al., 2021). In addition, the study reported by Vivarelli et al. did not include a treatment arm involving exposure to conventional cigarette mainstream smoke (Vivarelli et al., 2021).\n\nFurther independent in vivo studies into the toxicological and histopathological effects of heated tobacco products may be warranted as well as the development of standardized methods for the in vivo toxicological assessment of heated tobacco products. A full assessment of biomarkers of exposure should be conducted in in vivo studies to ensure comparable exposure between treatment arms. In addition, it should be noted that only one currently commercially available heated tobacco product (THS 2.2/IQOS) has been investigated in in vivo studies and no data exists for other commercially available heated tobacco products at this time. It is important to note however that the relevance of in vivo studies for assessing the effects of heated tobacco product aerosols for human exposure Is unclear. The strongest evidence for the assessment of heated tobacco products is likely to be derived from actual health outcomes in cohorts of heated tobacco product users compared to cohorts of smokers and non-smokers.\n\n\nIn vitro toxicology\n\nThis section of the review will discuss those studies that have investigated the in vitro toxicological effects of heated tobacco products. These studies have investigated the toxicological properties of whole aerosol produced from heated tobacco products as well as that of total particulate matter (produced by passing the aerosol through a Cambridge filter pad), the gas-vapour phase and extracts produced by dissolving the total particulate matter in a range of solvents including water. Several of the methods used in these studies have been validated specifically for use with heated tobacco products (Buratto et al., 2018; Bozhilova et al., 2020).\n\nCORESTA recommends three toxicology tests for the in vitro toxicological testing of tobacco smoke (CORESTA, 2004):\n\n1. Ames Salmonella typhimurium test (bacterial mutagenicity assay),\n\n2. Micronucleus assay and mouse lymphoma assay (mammalian genotoxicity assays),\n\n3. Neutral Red Uptake assay (mammalian cytotoxicity assay)\n\nWith respect to the Ames Salmonella typhimurium test [also known as the Ames test], several heated tobacco products including the EHCSS, EHCSS Series K, THS 2.2 and THP 1.0 have been assessed using this assay (Tewes et al., 2003; Roemer et al., 2004, 2008; Werley et al., 2008; Zenzen et al., 2012; Schaller et al., 2016a; Breheny et al., 2017; Crooks et al., 2018; Thorne et al., 2018; Le Godec et al., 2019; Wang H et al., 2021). The results from these studies indicate that total particulate matter, the gas/vapour phase and/or whole aerosol produced from these heated tobacco products either demonstrated an absence of mutagenicity under test conditions or a significant reduction in mutagenicity when compared to conventional cigarettes. There are no studies available which suggest that heated tobacco products have either comparable or greater mutagenicity than conventional cigarettes in the Ames test. These observations are in line with an earlier experimental study in which tobacco tablets were heated to temperatures of between 250°C and 550°C, and which concluded that the temperature to which the tobacco was heated had a significant effect on the subsequent mutagenicity of the aerosol produced from the tobacco tablets (White et al., 2001). No bacterial mutagenicity was detected in this study for temperatures of between 250°C and 360°C for strain TA98 and for temperatures of between 250°C and 400°C for strain TA100.\n\nWith respect to those mammalian assays which are used to investigate cytogenetics/mutation [micronucleus assay and mouse lymphoma assay], several heated tobacco products including the EHCSS, EHCSS Series K, THS 2.2 and THP 1.0 have been assessed using these assays (Schramke et al., 2006; Roemer et al., 2008; Werley et al., 2008; Schaller et al., 2016a; Crooks et al., 2018; Thorne et al., 2018, 2019a, 2019b, 2020; Le Godec et al., 2019; Wang H et al., 2021). The results from these studies indicated that total particulate matter, the gas/vapour phase and/or whole aerosol produced from heated tobacco products either demonstrate an absence of genotoxicity under the conditions of test or a significant reduction in genotoxicity when compared to conventional cigarettes in both assays. There are no studies available in the scientific literature which suggest that heated tobacco products have either comparable or greater genotoxicity than conventional cigarettes in either of the discussed assays.\n\nWith respect to those assays which are used to investigate cytotoxicity (Neutral Red uptake assay), several heated tobacco products including the EHCSS, EHCSS Series K, THS 2.2 and THP 1.0 have been assessed using this assay (Tewes et al., 2003; Roemer et al., 2004, 2008; Werley et al., 2008; Zenzen et al., 2012; Schaller et al., 2016a; Breheny et al., 2017; Adamson et al., 2018; Crooks et al., 2018; Jaunky et al., 2018; Leigh et al., 2018b; Murphy et al., 2018; Thorne et al., 2018; Davis et al., 2019a; Caruso et al., 2021). The results from these studies indicated that total particulate matter, the gas/vapour phase and/or whole aerosol produced from heated tobacco products typically demonstrate a significant reduction in cytotoxicity when compared to conventional cigarettes, with five studies reporting comparable cytotoxicity for some, but not all, of their experimental analyses. Only a single study has reported increased cytotoxicity for heated tobacco products compared to conventional cigarettes (Zenzen et al., 2012). In this study, three different variants of the heated tobacco product [which is no longer commercially available] were compared with a range of different commercially available conventional cigarettes ranging in stated tar yields including two 1 mg products.\n\nCytotoxicity, as assessed using in vitro assays other than the Neutral Red Assay, has also been shown to be absent for experimental reagents produced from heated tobacco products (Ito et al., 2019; Davis et al., 2019a; Bishop et al., 2020), present at lower levels than conventional cigarettes (Munakata et al., 2018; Bozhilova et al., 2020) or present at comparable levels to conventional cigarettes (Davis et al., 2019a).\n\nThere are many studies, published both by heated tobacco product manufacturers and by independent researchers, which have discussed the in vitro toxicological effects of heated tobacco products using a range of assays other than the CORESTA recommended assays.\n\nWith respect to those published by heated tobacco product manufacturers, all have concluded that exposure to aerosols and/or extracts produced from heated tobacco products resulted in in vitro toxicological effects which were significantly reduced compared to those effects seen after treatment with conventional cigarette mainstream smoke and/or extracts.\n\nUsing human coronary arterial endothelial and THP-1 monocytic cells, chemotaxis and transendothelial migration has been assessed after exposure to aerosol extracts produced from the THS2.2 heated tobacco product as a surrogate for early pathological stages of atherosclerosis (Van der Toorn et al., 2015). Extracts produced from the THS2.2 heated tobacco products demonstrated a reduced effect, with extracts produced from the 3R4F Kentucky reference cigarette being approximately 18 times more potent than those produced from the THS2.2 heated tobacco product. A subsequent study quantified adhesion of monocytic cells to human coronary arterial endothelial cells after exposure to extracts produced from both 3R4F Kentucky reference cigarette and the THS2.2 heated tobacco product as an experimental surrogate for the initiation of atherogenesis (Poussin et al., 2016). The THS2.2 heated tobacco product displayed a reduced effect in both fresh direct and indirect exposure studies compared to the reference cigarette. Comparable results were observed with the THP1.0 heated tobacco product where no significant inhibition in wound healing was observed using aqueous extracts from the heated tobacco product in an endothelial migration assay (Bishop et al., 2020). Further studies investigated oxidative stress and inflammatory end-points in human bronchial epithelial cells after exposure to total particulate matter from both the THP1.0 and THS2.2 heated tobacco products and the 3R4F Kentucky reference cigarette (Taylor et al., 2018; van der Toorn et al., 2018). Using a luciferase-based reporter assay, transcriptional activation of the antioxidant response elements (ARE) was assessed 6 and 24 hours after exposure to the aerosols (Taylor et al., 2018). Using high-content screening (HCS), ten different toxicity and oxidative stress endpoints (ATP, cell count, glutathione content, mitochondrial mass, mitochondrial membrane potential, nuclear size, reactive oxygen species formation, DNA structure, DNA damage and c-Jun stress kinase) were assessed four and twenty-four hours after exposure. Exposure to total particulate matter from the 3R4F Kentucky reference cigarette after either 6- or 24-hours induced a significantly greater activation of the ARE than either of the heated tobacco products across all doses tested. Neither of the heated tobacco products elicited any responses in the ten HCS end-points at either time point, whilst the 3R4F Kentucky Reference cigarette caused statistically significant perturbations in four of the end-points (ATP, glutathione content, mitochondrial membrane potential and DNA damage) after 4 and/or 24 hours of exposure. The second study demonstrated that exposure of human bronchial epithelial cells (BEAS-2B) over a 12-week period to total particulate matter produced from the THS2.2 heated tobacco product did not produce an increase in inflammatory mediators at a five-fold higher concentration when compared to total particulate matter from the 3R4F Kentucky reference cigarette (van der Toorn et al., 2018).\n\nAerosol from the THS2.2 heated tobacco product has been demonstrated to have no quantifiable inhibitory effect on monoamine oxidase activity (whilst mainstream smoke from the 3R4F Kentucky reference displayed a significant inhibitory effect) (van der Toorn et al., 2019) whilst separate studies have indicated that total particulate matter from the THS2.2 heated tobacco product produced a markedly lower effect on mitochondrial dynamics and biogenesis than total particulate matter from the 3R4F Kentucky reference cigarette (Malińska et al., 2018; Walczak et al., 2020). A dose of 150μg/ml TPM from the THS2.2 heated tobacco product was required to achieve the same effects on mitochondrial end-points as a dose of 7.5μg/ml TPM from the 3R4F Kentucky reference cigarette (twenty times higher dose) (Walczak et al., 2020).\n\nAnalysis of the transcriptomic changes (based on RNA sequencing) in 3D human airway cells due to exposure to experimentally-generated aerosols from the THP1.0 and THS2.2 heated tobacco products suggested that the THP1.0 heated tobacco product had a markedly lower transcriptomic effect compared to the THS2.2 heated tobacco product (Haswell et al., 2018). A further study employing a 3D model of goblet cell hyperplasia found that a six-week exposure period using whole smoke from the 3R4F Kentucky reference cigarette demonstrated a significant decrease in transepithelial electrical resistance and an increase in MUC5AC positive cells (cells expressing mucin) whilst exposure to aerosol from the THP1.0 did not (Haswell et al., 2021).\n\nSystems Toxicology Assessments conducted by PMI have indicated a significantly lower and more transient response in terms of gene, microRNA and/or protein expression in cell lines as a result of exposure to experimentally generated aerosols or aqueous extracts from the THS2.2/IQOS heated tobacco product compared to comparable treatments produced from the 3R4F Kentucky Reference cigarette. Exposure to aerosols and/or extracts was conducted using comparable nicotine concentrations with several concentrations used for either or both of the exposure treatments. These studies have used the adhesion of monocytic cells to human coronary endothelial cells as a surrogate pathophysiologically relevant event in atherogenesis (Poussin et al., 2016, 2020), 3D nasal epithelial culture models (Iskandar et al., 2017a), organotypic nasal epithelial tissue cultures (Iskandar et al., 2017c), organotypic oral epithelial tissue cultures (Zanetti et al., 2016), organotypic buccal epithelial tissue cultures (Iskandar et al., 2017c), organotypic bronchial epithelial tissue cultures (Iskandar et al., 2017b, 2017c), organotypic gingival epithelial tissue cultures (Zanetti et al., 2017), small airway epithelium models (Iskandar et al., 2017d) and aortic smooth muscle cells (Poussin et al., 2021). A meta-analysis on the relative expression of microRNA using data collected from twelve in vitro studies investigating conventional cigarettes and potentially reduced risk products (including heated tobacco products) determined a 94% reduction in microRNA expression relative to conventional cigarette exposure (Sewer et al., 2020). No specific microRNA expression response pattern could be identified after heated tobacco product exposure.\n\nWith respect to those studies published by independent researchers, most, but not all, have concluded that exposure to aerosols and/or extracts produced from heated tobacco products produced in vitro toxicological effects which were comparable to those effects seen after treatment with conventional cigarette mainstream smoke and/or extracts produced from it.\n\nWith respect to those studies that have found comparable effects, one of the first studies published by independent researchers reported that the IQOS heated tobacco product demonstrated comparable effects to a conventional cigarette (Marlboro Red) on cell cytotoxicity as assessed using the MTT and lactate dehydrogenase assays (Sohal et al., 2019; McAlinden et al., 2019, 2020). A subsequent study also using the IQOS heated tobacco product reported a comparable oxidative stress response in primary rat alveolar epithelial cells after exposure to extracts produced from both the IQOS heated tobacco product and the Marlboro Red conventional cigarette (Ito et al., 2020). Assessment of oxidative stress by quantification of intracellular oxidised and reduced glutathione in bronchial epithelial cells (BEAS-2B) by separate authors reached comparable conclusions albeit after more intensive exposure conditions for the heated tobacco product under investigation (IQOS) (Dusautoir et al., 2021) whilst a second research group reported that changes in intracellular glutathione were significantly less pronounced for the IQOS heated tobacco product than for either a commercially available conventional cigarette or the 3R4F Kentucky Reference cigarette (Wang L et al., 2020). Exposure of Jurkat T cells to either filtered air, conventional cigarette mainstream smoke or aerosol from the IQOS heated tobacco product for a thirty-minute period demonstrated a statistically significant decrease in cell viability, an increase in necrotic cells and a greater percentage of early (but not late) apoptotic cells but no statically significant increases in oxygen and nitrogen reactive species and oxidative DNA damage (Scharf et al., 2021).\n\nIn opposition to these studies, several studies published by independent researchers have found no significant in vitro toxicological effects. A study investigating effects on adipocytes demonstrated no statistically significant effect on pre-adipocyte survival and differentiation to beige adipocyte after treatment with extracts produced from the IQOS heated tobacco products whilst extracts produced from mainstream smoke from the 1R6F Kentucky reference displayed a significant decrease in cell viability in a dose-dependent fashion (Zagoriti et al., 2020). Investigations into the effects of the IQOS heated tobacco product on osteoprogenitor cell viability and function found that aqueous extracts produced from the IQOS heated product were significantly less toxic to bone than aqueous extracts produced from Marlboro conventional cigarettes when analysed by mitochondrial and esterase activity (Aspera-Werz et al., 2020). Significant effects from treatment with the IQOS heated tobacco product extracts were only observed at very high, non-physiologically relevant concentrations whilst extracts from the Marlboro conventional cigarette significantly reduced alkaline phosphatase activity and matrix mineralisation at low concentrations. A study investigating cell viability in human gingival fibroblasts and human keratinocytes using the MTT assay found that extracts produced from the IQOS heated tobacco products increased both cell viability (23% to 41% with fibroblasts and 30% to 79% with keratinocytes) and cell migration (Pagano et al., 2021). IQOS extracts were not reported to induce an increase in cell death or produce morphological alterations although an increase in p53 expression in fibroblasts and a decrease in p53 expression in keratinocytes was observed. Exposure to total particulate matter produced from the MOK heated tobacco product (produced by the China Tobacco Hubei Industrial Company) and the 3R4F Kentucky Reference cigarette in human lung cancer cells (NCI-H292) showed that whilst the 3R4F Kentucky Reference cigarette demonstrated dose-dependent responses in the majority of the fifteen-toxicity end-points investigated, exposure to comparable doses produced from the MOK heated tobacco product did not (Wang H et al., 2021).\n\nA small number of in vitro studies have investigated the effect of heated tobacco products on the discolouration of dental resin composites (Zhao et al., 2017), extracted bovine enamel preparations (Dalrymple et al., 2018; Haiduc et al., 2020; Dalrymple et al., 2021), extracted human teeth prepared with composite resin restorations (Zanetti et al., 2019) and artificial denture teeth (Wang Y et al., 2021) as experimental surrogates to assess whether or not heated tobacco product use has the potential to discolour the teeth of users. Experimentally produced samples were exposed to the particulate matter and/or aerosol produced from the THS2.2/IQOS or THP1.0 heated tobacco products and compared with samples exposed to conventional cigarette smoke. Sample colour was assessed quantitatively using spectrophotometric assessment. In each instance, the heated tobacco product under investigation was found to result in significantly less discolouration to the dental resin composites, extracted bovine enamel preparations, human teeth or artificial denture teeth than the smoke from conventional cigarettes.\n\nGiven the data available in the scientific literature on the fewer and substantially lower levels of HPHCs present in the aerosols experimentally produced from heated tobacco products, it is possible to perform risk assessment modelling of the overall harm from their use and compare this with the harm estimated to be associated with conventional cigarette use (Stephens, 2018; Lachenmeier et al., 2018; Slob et al., 2020; Rodrigo et al., 2021). This approach takes into consideration the different toxicological effects of each of the chemical constituents analysed and attempts to provide an overall estimate of harm.\n\nUsing data from a single study (Schaller et al., 2016b), the first study attempted to estimate the cancer potencies of various product types using levels of chemical constituents found in the aerosols of each product type and their associated inhalation unit risks (Stephens, 2018). Using this approach, the author estimated that the heated tobacco product under investigation had a lower cancer potency (compared to that of conventional cigarettes) by at least one order of magnitude but a higher cancer potency than non-tobacco containing vapour products and a nicotine inhaler.\n\nUsing a margin of exposure (MOE) approach, subsequent authors attempted to conduct a quantitative risk assessment (Lachenmeier et al., 2018). The MOE was defined as the ratio between the toxicological threshold of the chemical constituent and the estimated human intake of the same chemical constituent (with a higher MOE indicating a lower risk). This study noted the observations of the prior study of Auer et al. (2017a), which suggested that levels of acenaphthene were found to be higher in heated tobacco products than in conventional cigarettes. Lachenmeier et al. found that, with the exception of acenaphthene, the MOE of several chemical constituents (acetaldehyde, ammonia, arsenic, chromium, catechol, formaldehyde and pyridine) were increased by factors of between three and nine for heated tobacco products, compared to conventional cigarettes and for the rest for the chemical constituents exceeded a factor of ten up to a factor of 415 for isoprene. For several chemical constituents (acenaphthene, benzene, chromium, m/p-cresol, ethylene oxide, isoprene, NNN, NNK, quinoline and styrene), the MOE in heated tobacco products exceeded the threshold of 10,000 reported by the authors as being the threshold for genotoxic carcinogens. For conventional cigarettes, only acenaphthene exceeded this threshold. Overall, the results of this study indicated that the combined MOEs for all chemical constituents analysed (including acenaphthene) was twenty-three-fold higher for heated tobacco products compared to conventional cigarette use in the absence of nicotine and ten-fold (one order of magnitude) higher compared to conventional cigarette use with nicotine present. Furthermore, the study indicated that acenaphthene, present either in conventional cigarette smoke or in aerosol produced from heated tobacco products, is considered unlikely to pose any significant health risk to consumers from either product type use due to its MOE exceeding 10,000 for both conventional cigarettes and heated tobacco products.\n\nUsing an approach which focuses on the changes in cumulative exposure for a defined number of specific chemical constituents present in both the aerosol from heated tobacco products and mainstream smoke from conventional cigarettes, separate authors have attempted to compare the carcinogenicity of both product types (Slob et al., 2020). Using data from a single experimental study (Schaller et al., 2016b) which reported levels of eight known or suspected carcinogens (acrylonitrile, acetaldehyde, 1,3-butadiene, ethylene oxide, formaldehyde, benzo [a] pyrene, nitrobenzene and propylene oxide) in both the aerosol from heated tobacco products and mainstream smoke from conventional cigarettes, the changes in cumulative exposure was estimated to be between ten and twenty-five times lower when using heated tobacco products compared to conventional cigarettes. The authors concluded that “such a change indicates a substantially smaller reduction in expected life span based on available dose-response information in smokers”. Despite this conclusion, it was noted that “an unfavourable health impact related to heated tobacco products remains as compared to complete abstinence”. It should be noted that the findings of this study are limited by the fact that only eight chemical constituents were investigated.\n\nThe most recently published study estimated both cancer potency and mean lifetime cancer risk for HPHCs present in heated tobacco product aerosol and used an MOE approach for non-carcinogenic HPHCs (Rodrigo et al., 2021). The authors used experimentally-determined HPHC data obtained from commercially available HTPs (n=8) and conventional cigarettes (n=273) in the estimations. A total of forty-two and thirty-three HPHCs were quantified in heated tobacco product aerosol and conventional cigarette mainstream smoke respectively. Cancer potency was determined for each selected HPHC and translated to mean lifetime cancer risk for each evaluated product. Mean lifetime cancer risk values were estimated for conventional cigarettes and heated tobacco products with a median value of 2.73×10-2 (range of 1.40×10-2 to 3.97×10-2) for conventional cigarettes and a median value of 1.106×10-3 (range of 4.53×10-5 to 3.95×10-3) for heated tobacco products. Based on their median values, the authors reported that the relative cancer risk for a lifetime exposure to heated tobacco products was 0.039 when compared to conventional cigarettes. Total MOE values (including that for nicotine) for conventional cigarettes had a median value of 1.36×10-4 (range of 1.03×10-4 to 2.16×10-4) whilst the median value for heated tobacco products was 4.49×10-3 (range of 1.40×10-3 to 1.42×10-2). An increase in the median value for heated tobacco products was reported to indicate an estimated reduced non-cancer risk compared to conventional cigarette use.\n\nThere appears to be a significant disparity in regard to the reported findings of in vitro studies investigating heated tobacco products. Those studies published by heated tobacco product manufacturers have indicated that exposure to aerosol, particulate matter, gaseous phase components or extracts experimentally produced from heated tobacco products consistently yielded significantly reduced toxicological effects when compared to those observed with conventional cigarettes in a range of cell lines including those derived from the respiratory tract and aerodigestive tracts across a range of different assays. In many instances, a several-fold/several order of magnitude reduction in biological effect was observed between conventional cigarettes and heated tobacco products. In opposition, a significant proportion of those number of studies published by independent researchers have concluded that exposure to aerosol, particulate matter, gaseous phase components or extracts experimentally produced from heated tobacco products produced comparable effects to those seen with conventional cigarettes. This disparity may be due to the use of different cell line exposure methodologies, and/or the use of different methods to generate heated tobacco product aerosol and conventional cigarette mainstream smoke and differences in data collection and data treatment. Further research is required in regard to the comparison between relative and absolute risk associated with heated tobacco product use (i.e. comparing differences between changes in single constituents and changes in cumulative levels). It should be noted, however, that heated tobacco products have been consistently demonstrated to have decreased mutagenic, genotoxic and cytotoxic effects when compared to conventional cigarettes in those in vitro assays recommended by CORESTA for use with tobacco smoke.\n\nThese conclusions are in agreement with those of a separate review published by PMI researchers, which concluded that “the experimental results [from systems biology and systems toxicology studies] demonstrate a reduced impact on apical and molecular endpoints, no novel effect not seen with cigarette smoke exposure, and an effect of switching from cigarettes to either MRTP [two heated tobacco products; one electrically heated and one with a carbon-heating design] that is comparable to that of complete smoking cessation” (Schlage et al., 2020). PMI researchers have also attempted to further validate their scientific approach to assessment of the THS2.2 heated tobacco product by providing their biological and clinical samples or data packages to external independent researchers (in a blinded fashion) to see whether or not they would reach the same conclusions as the original researchers (Poussin et al., 2017; Boué et al., 2019; Belcastro et al., 2020). In each instance reported, the external researchers corroborated with the conclusions reached by the original researchers based on their own independent analyses of the samples.\n\n\nNicotine pharmacokinetics and pharmacodynamics\n\nThis section of the review will detail those studies that have investigated the pharmacokinetic and pharmacodynamic profile of nicotine delivery associated with the use of heated tobacco products under controlled clinical conditions.\n\nFour controlled clinical studies involving adult smokers have been published which have attempted to estimate the pharmacokinetic profile of nicotine delivery from heated tobacco products and compare this delivery with that observed with conventional cigarettes. Two studies have been published by PMI (Picavet et al., 2016; Brossard et al., 2017) whilst two studies have been published by independent researchers (Maloney et al., 2021; Phillips-Waller et al., 2021).\n\nWith respect to those studies published by PMI, the first study was conducted using 28 adult smokers and investigated nicotine pharmacokinetics after single and ad libitum use of the THS 2.1 heated tobacco product and the subjects’ own brand of conventional cigarettes during a 7-day clinical confinement period (Picavet et al., 2016). The median time to maximum nicotine concentration (Tmax) was eight minutes after single use of both products. The median time to the peak nicotine concentration following ad libitum use was similar for both product types. The average maximum plasma nicotine concentration (Cmax) after single use of the heated tobacco product was 8.4ng/ml, equivalent to approximately 70% of that obtained with the subjects’ own brand of conventional cigarettes. A transient reduction from baseline in the urge to smoke of 40% was observed 15 minutes after single use of both product types whilst the mean Questionnaire of Smoking Urges (QSU-brief) total scores following both single and ad libitum use were similar for both product types. The second study was conducted with Japanese adult smokers using both mentholated and non-mentholated variants of the THS 2.2 heated tobacco product, subjects’ own brand of conventional cigarettes and nicotine gum indicated that maximal nicotine concentration, overall nicotine exposure and urge-to-smoke scores were similar between the heated tobacco product and the conventional cigarette (Brossard et al., 2017). Maximal nicotine concentration (Cmax) and area under the curve from the start of product use to time of last quantifiable concentration (AUC0-last) were comparable between the mentholated and non-mentholated variants of the heated tobacco product and conventional cigarettes with ratios varying between 88 and 104% for Cmax and from 96 to 98% for AUC0-last. Pharmacokinetic profiles were largely comparable between the mentholated and non-mentholated variants of the heated tobacco product, although the mentholated variant did show a slightly lower Cmax (10.70ng/ml) than the non-mentholated variant (14.30ng/ml).\n\nWith respect to those studies published by independent researchers, the first study assessed nicotine delivery and subjective effects in 18 adult smokers after controlled (10 puffs with a 30-second inter-puff interval) or ad libitum (90-minute session) use of the IQOS heated tobacco product, JUUL e-vapour product or the subjects’ own brand of conventional cigarettes (Maloney et al., 2021). The subjects had no prior experience of the IQOS or JUUL products. Use of the IQOS heated tobacco product increased mean plasma nicotine levels from 2.1±0.2ng/ml to 12.7±6.2ng/ml after 10 puffs and to 11.3±8.0ng/ml after ad libitum use. Use of the JUUL e-vapour product increased mean plasma nicotine levels from 2.2±0.7ng/ml to 9.8±4.9ng/ml after 10 puffs and to 11.5±9.3ng/ml after ad libitum use. Use of the subjects’ own brand of conventional cigarettes increased mean plasma nicotine levels from 2.1±0.2ng/ml to 20.4±1.4ng/ml after 10 puffs and to 21.0±10.2ng/ml after ad libitum use. Mean plasma nicotine levels were significantly higher after use of the subjects’ own brand of conventional cigarettes when compared to use of either IQOS or JUUL (p<0.05) although no information was provided as to whether or not there was a statistically significant difference between IQOS and JUUL use. Subjective measures of product use, determined using the QSU-brief and Visual Analogue Scales, indicated that nicotine cravings and urges to smoked were reduced significantly for all products investigated after controlled product use and for subjects’ own brand of conventional cigarettes and the IQOS heated tobacco product after ad libitum use. The second study used a comparable study design to assess the pharmacokinetic profile of nicotine delivery after 5 minutes of ad libitum use of subjects’ own brand of conventional cigarettes, the IQOS heated tobacco product, the JUUL e-vapour product and a separate refillable vaping product (Phillips-Waller et al., 2021). The study involved 22 daily e-vapour users who smoked less than one conventional cigarette a day and who had undergone overnight abstinence from smoking and e-vapour use prior to the experimental sessions. A baseline blood sample was obtained prior to product use and additional samples taken 2, 4, 6, 10 and 30 minutes after starting product use. Results indicated that use of the IQOS heated tobacco product delivered about half as much nicotine over the 30-minute period with a similar number of puffs than use of the subjects’ own brand of conventional cigarette (median Cmax, median Tmax and median AUC0 to 30 values for IQOS and subjects’ own brand of conventional cigarette were 8.3ng/ml, 4.0 minutes and 152.0 and 12.9ng/ml, 6.0 minutes and 314.7 respectively). Use of the IQOS heated tobacco product also had a lower median Cmax and lower median AUC0 to 30 than use of the JUUL e-vapour product (Cmax of 19.6ng/ml and AUC0 to 30 of 343.2) whilst the median Tmax was the same (4.0 minutes).\n\nA key requisite for any heated tobacco product is that it provides a satisfying alternative to adult smokers who would otherwise continue to smoke. A heated tobacco product with a comparable nicotine uptake profile to conventional cigarettes is likely to be more satisfying to more adult smokers considering heated tobacco products as an alternative to continued cigarette smoking. The available clinical evidence indicates that use of heated tobacco products provides a nicotine pharmacokinetic profile comparable to, or marginally less than, that observed with conventional cigarette use dependent on the study design employed and pharmacokinetic parameters quantified.\n\nThese findings are in agreement with the conclusions of a separately published review on nicotine pharmacokinetics associated with use of heated tobacco products (Marchand et al., 2017). This review, written by PMI researchers, compared the nicotine pharmacokinetics of the THS heated tobacco product with that observed with conventional cigarettes and two nicotine replacement therapy products (nicotine nasal spray and oral nicotine gum) based on data obtained from eight PMI-sponsored clinical trials (Marchand et al., 2017). The review concluded that background-adjusted exposure to nicotine was consistently lower with the THS heated tobacco product compared to that observed with conventional cigarettes by an average of 24% or 26% depending on whether the results were based on Cmax or area under the curve (AUC).\n\n\nBiomarkers\n\nThis section of the review will detail those studies that have quantified levels of biomarkers of exposure and/or biomarkers of potential harm associated with the use of heated tobacco products in clinical assessments.\n\nAcute exposure studies\n\nNine acute exposure studies are available that describe the effects of controlled use of heated tobacco products on exhaled carbon monoxide levels (Buchhalter and Eissenberg, 2000; Buchhalter et al., 2001; Breland et al., 2002; Adriaens et al., 2018; Caponnetto et al., 2018; Nga et al., 2020; Pataka et al., 2020; Ikonomidis et al., 2021; Maloney et al., 2021). Five studies reported no statistically significant increase in observed levels of exhaled carbon monoxide after controlled use of a heated tobacco product whilst reporting a statistically significant increase in observed levels after controlled use of conventional cigarettes (Buchhalter and Eissenberg, 2000; Buchhalter et al., 2001; Caponnetto et al., 2018; Ikonomidis et al., 2021; Maloney et al., 2021). The other four studies reported minimal, but statistically significant, increases in exhaled carbon monoxide levels when compared to baseline levels with heated tobacco product use (Breland et al., 2002; Adriaens et al., 2018; Nga et al., 2020; Pataka et al., 2020). In eight of the nine studies, at no time-point analysed did the exhaled carbon monoxide levels exceed 10ppm during and/or after heated tobacco product use, with this value being commonly accepted as the cut-off value for the reference range of non-smokers (Caponnetto et al., 2018). In the remaining study, inclusion criteria for the conventional cigarette smokers required an exhaled carbon monoxide levels exceeding 10ppm prior to conducting the study (Ikonomidis et al., 2021).\n\nThese findings are in line with those of a separate study which assessed the effect of increasing use of the Accord heated tobacco product on exhaled carbon monoxide levels (and number of cigarettes smoked per day) in eleven conventional cigarette smokers who were required to use increasing amounts of Accord (5, 10 and 15 sticks per day) whilst being allowed to continue to use their existing cigarette (Hughes and Keely, 2004). Use of the heated tobacco product decreased the number of conventional cigarettes smoked per day and exhaled carbon monoxide levels in dose-dependent manner. Using Accord fifteen times per day decreased the number of conventional cigarettes smoked by 32% (-8.6 cigarettes per day) and exhaled carbon monoxide levels by 27% (-5.9ppm).\n\nForced switching studies\n\nTwenty-one forced switching studies are available which describe a study protocol involving volunteers within closed clinical settings and/or in “real-world” [also known as “ambulatory”] scenarios. Table 3 details the heated tobacco product(s) investigated, the number of study participants and switching period used and the biomarker(s) of exposure quantified in each study. A total of twenty-four separate biomarkers of exposure have been quantified across the reported studies. The volunteers involved in these studies were adult conventional cigarette smokers typically with a significant self-reported smoking history. In the majority of studies, the volunteers are brought into a closed clinical setting and allowed to settle for several days before being randomised into one of several specific groups for a period of five to eight days. One group continue to smoke conventional cigarettes [either brought into the clinic by the volunteers themselves or provided to them by the study staff], another group are switched to the heated tobacco product under investigation, whilst a final group (if present) do not use any tobacco- or nicotine-containing products (and act as the smoking abstinence/control group). Product consumption is controlled by the study staff to ensure approximately comparable consumption (in terms of number of sticks smoked per day) between the two product groups and typically takes into account the prior self-reported smoking history of the volunteers. Two of the twenty-one studies included an additional period of “real-world” use following the initial period of clinical confinement whilst five of the twenty-one studies did not include any period of clinical confinement and solely investigated “real-world” use with this approach reported to offer a more realistic method of assessing product use by consumers. The results shown in Table 3 indicate whether the quantified biomarker of exposure was determined to be at a significantly lower level following heated tobacco product use at the end of the clinical confinement period when compared to conventional cigarette use (as indicated by a downwards arrow and green shading) or whether observed levels were comparable with use of both product types (as indicated by an equivalency arrow and orange shading). The aim of this approach is to demonstrate that switching to the heated tobacco product(s) under investigation from conventional cigarettes resulted in a quantifiable, and statistically significant, decrease in levels of biomarkers of exposure to select toxicants associated with cigarette smoking whilst maintaining comparable cigarette consumption between the two product use groups.\n\nIf the quantified biomarker of exposure was determined at the end of the clinical confinement period to be at a significantly lower level following heated tobacco product use when compared to conventional cigarette use, then this is indicated by a downwards arrow and green shading; if the quantified levels were comparable, with no statistically significant difference, after use of both product types, then this is indicated by an equivalency arrow and orange shading. 1-NA, 1-aminonaphtalene; 2-NA, 2-aminonaphthalene; 1-OHP, 1-hydroxypyrene; 3-HMPMA, 3-hydroxy-1-methylpropylmercapturic acid; 3-HPMA, 3-hydroxypropylmercapturic acid; 3-OH-B [a] P, 3-hydroxy-benzo [a]pyrene; 4-ABP, 4-aminobiphenyl; AAMA, N-acetyl-S-(2-carbamoylethyl)cysteine; B [a] P, benzo [a]pyrene; CEMA, 2-cyanoethylmercapturic acid; COHb, carboxyhaemoglobin; EHCSS, Electrically Heated Cigarette Smoking System; eCO, exhaled carbon monoxide; GAMA, N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine; HEMA, 2-hydroxyethylmercapturic acid; MHBMA, monohydroxybutenyl mercapturic acid; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNN, N-nitrosonornicotine; S-PMA, S-phenylmercapturic acid; TMA, trans, trans-muconic acid.\n\nThe results from these studies, as shown in Table 3, indicate that use of those heated tobacco products which are currently commercially available (glo and IQOS) under highly defined and controlled conditions results in statistically significant reductions in quantified levels of all biomarkers of exposure when compared to those observed with continued use of conventional cigarettes under the same experimental and clinical conditions. In six of the twenty studies, several biomarkers of exposure associated with nicotine were found to be comparable between the two product types indicating a comparable nicotine delivery with the heated tobacco product (IQOS) and conventional cigarette under investigation.\n\nThese findings are in agreement with the conclusions of two published systematic reviews on the levels of biomarkers of exposure associated with use of heated tobacco products (Drovandi et al., 2020; Akiyama and Sherwood, 2021). The first systematic review identified ten randomised controlled trials involving 1,766 participants published between January 2010 and August 2019 which quantified twelve biomarkers of exposure after controlled use of both heated tobacco products and conventional cigarettes (Drovandi et al., 2020). Seven of these ten studies are detailed in Table 3 whilst the other three articles related to either use of a hybrid product (n=2) or a carbon-heated tobacco product (n=1) both of which are outside the remit of this review. The authors of this systematic review concluded that “in comparison to conventional cigarettes, all twelve biomarkers of exposure assessed were significantly lower for participants assigned to a heat-not-burn device. In comparison to smoking abstinence, heat-not-burn devices were statistically equivalent for eight biomarkers of exposure and significantly elevated for four biomarkers of exposure” (Drovandi et al., 2020). The second systematic review identified twenty-five randomised controlled trials published up to April 2020 which quantified twelve biomarkers of exposure after controlled use of both heated tobacco products and conventional cigarettes (Akiyama and Sherwood, 2021). Nineteen of these twenty-five studies are detailed in Table 3 whilst the other six articles related to either use of a hybrid product (n=2) or a carbon-heated tobacco product (n=4). The authors of this systematic review concluded that “taken together, all findings suggest that biomarker of exposure levels in users of e-cigarettes and heated tobacco products show a significant reduction compared to a cigarette condition (or cigarette baseline)” (Akiyama and Sherwood, 2021).\n\nSurvey cohort/smoking cessation studies\n\nA smoking cessation study demonstrated statistically significant decreases in both exhaled carbon monoxide and blood carboxyhaemoglobin levels in forty male adult conventional cigarette smokers who switched to either a heated tobacco product (THS 2.2) or an e-vapour product from their own brand of conventional cigarettes for a period of six months (n=20 in each group) (Beatrice and Massaro, 2019). Levels of both exhaled carbon monoxide and blood carboxyhaemoglobin with heated tobacco product use were reported as being within the range associated with non-smokers.\n\nA study of 182 Japanese men aged between eighteen and sixty-four provided urinary biomarker data in relation to heated tobacco product use (Kawasaki et al., 2021). A lifestyle questionnaire was given to the participants as well as collection of a urine sample. Twenty-two men self-reported heated tobacco product use whilst forty-eight men self-reported conventional cigarette use. No information was provided with respect to duration or intensity of product use or incidence of dual product use. Urinary nicotine and cotinine levels did not show a statistically significant difference between heated tobacco product users and conventional cigarette smokers whilst levels of 3’-hydroxycotinine, total nicotine equivalents and NNAL were higher in conventional cigarette smokers when compared to heated tobacco product users (p<0.05 for 3’-hydroxycotinine and total nicotine equivalents and p<0.01 for NNAL).\n\nA subsequent study investigated levels of six biomarkers of exposure (nicotine, cotinine, 3’-hydroxycotinine [metabolites of nicotine], NNAL [a metabolite of NNK], CEMA [a metabolite of acrolein] and CYMA [a metabolite of acrylonitrile]) in Korean adult smokers recruited into a cross-sectional study (Rudasingwa et al., 2021). Comparison of quantified levels for the six biomarkers of exposure between exclusive conventional cigarette smokers (n=403) and exclusive heated tobacco product users (n=76) showed that levels of nicotine, cotinine, 3’-hydroxycotinine and CYMA did not differ to a statistically significant extent between the user groups whilst levels of NNAL and CEMA were significantly lower in exclusive heated tobacco product users when compared to exclusive conventional cigarette smokers. Whilst the study did not provide quantitative data for dual product users, the authors did report that biomarker levels for dual users of heated tobacco products and conventional cigarette smokers were similar to those of exclusive conventional cigarette smokers.\n\nSeven of the forced switching studies previously discussed and detailed in Table 3 quantified levels of biomarkers of potential harm in addition to levels of biomarkers of exposure (Roethig et al., 2008; Martin Leroy et al., 2012; Lüdicke et al., 2018b, 2019; Haziza et al., 2020b; McEwan et al., 2021; Gale et al., 2021b). Table 4 details the heated tobacco product(s) investigated, the number of study participants and switching period used and the biomarkers of potential harm quantified in each study. The study protocol for a seventh forced switching study discussed and detailed in Table 3 indicated the intention of the authors to quantify levels of 8-epi-prostaglandin F2α and white blood count in the urine and whole blood of volunteers respectively (Gale et al., 2017). However, the published results from this study do not include any such data (Gale et al., 2019).\n\nIf the quantified biomarker of potential harm was determined at the end of the clinical confinement period to be at more clinically favourable levels following heated tobacco product use when compared to conventional cigarette use, then this is indicated by green shading; if the quantified levels were comparable, with no statistically significant difference, after use of both product types, then this is indicated by orange shading. 8-EPF, 8-epi-prostaglandin F2α; 11-DTX, 11-dehydrothromboxane B2; EHCSS, Electrically Heated Cigarette Smoking System; Hb, Haemoglobin; HDL-C, High Density Lipoprotein Cholesterol; hs-CRP, High Sensitivity C-Reactive Protein; LDL-C, Low Density Lipoprotein Cholesterol; RBC, Red Blood Cell; THS, Tobacco Heating System; WBC, White Blood Cell; BP, Blood Pressure; FENO, Fractional Exhaled Nitric Oxide; FEV1, Forced Expiratory Volume in one second; HB A1c, Haemoglobin A1c; sICAM-1, soluble Intracellular Adhesion Molecule-1.\n\nThe results shown in Table 4 indicate whether the quantified biomarker of potential harm demonstrated a statistically significant and physiologically favourable change with heated tobacco product use when compared to conventional cigarette use (as indicated by green shading) or whether no statistically significant physiological change was observed (as indicated by orange shading). It should be noted that unlike biomarkers of exposure, where a decrease in observed levels is invariably correlated to a decrease in exposure, physiologically favourable changes in biomarkers of potential harm may be associated with either a decrease or increase in their levels (such as with white blood cell count and high-density lipoprotein cholesterol respectively). Therefore, no arrows are included in Table 4 to avoid confusion. The aim of this approach is to demonstrate that switching to the heated tobacco product under investigation from conventional cigarettes resulted in a quantifiable, and statistically significant, physiologically favourable improvement in levels of biomarkers of potential harm whilst maintaining comparable product consumption between the two product use groups.\n\nThe results from these studies, as shown in Table 4, indicate that use of heated tobacco products results in statistically significant, and physiologically favourable improvements, in a small number of biomarkers of potential harm when compared to use of conventional cigarettes under the same experimental and clinical conditions. For the remaining biomarkers of potential harm, no significant differences between heated tobacco product use, conventional cigarette use and/or smoking abstinence were observed. The only biomarkers of potential harm which demonstrated physiologically favourable improvements with heated tobacco product use compared with conventional cigarette use across more than one of the six studies were white blood cell count (five studies), high-density lipoprotein cholesterol (three studies), 8-epi-prostaglandin F2α (four studies), 11-dehydrothromboxane B2 (two studies), haemoglobin (two studies), haematocrit (two studies) and soluble intracellular adhesion molecule-1 (two studies). For the two studies that also included an abstinence group who refrained use of any tobacco- and/or nicotine-containing products (Lüdicke et al., 2018b; Haziza et al., 2020b), only a very small number of biomarkers of potential harm demonstrated a statistically significant difference between this group and those who used the heated tobacco product: triglycerides, body weight and glucose (Lüdicke et al., 2018b) and white blood cell count (Haziza et al., 2020b). Researchers from PMI have also applied a systems pharmacology approach in an attempt to demonstrate a reduced exposure response in adult subjects who switched to the THS2.2 heated tobacco product from their regular conventional cigarette as part of a forced switching study (Haziza et al., 2016b) on a whole-blood based eleven gene response signature (Martin et al., 2016). A reduced exposure response was observed in subjects that either stopped smoking or switched to the THS2.2 heated tobacco product compared to subjects who continued to smoke their regular conventional cigarette. Comparable findings of a reduction in the same eleven gene response signature in subjects who either stopped smoking or switched to the THS2.2 heated tobacco product compared to those who continued to smoke conventional cigarettes were found when data was pooled from four separate forced switching studies separately published by PMI in six articles (Haziza et al., 2016a, 2016b, 2020a, 2020b; Lüdicke et al., 2018a, 2018b) using largely comparable designs (Martin et al., 2019).\n\nThese findings are in agreement with the conclusions of a separately published systematic review on the levels of biomarkers of potential harm associated with use of heated tobacco products (Akiyama and Sherwood, 2021), a pooled analysis of data from three clinical studies (Roethig et al., 2010) whilst in disagreement with the conclusions of a third author who published a review on the same topic (Glantz, 2018b). The systematic review identified seven randomised controlled trials published up to April 2020 which quantified biomarkers of potential harm after controlled use of both heated tobacco products and conventional cigarettes. Five of the seven studies are detailed in Table 4 whilst the other two studies related to use of carbon-heated tobacco products which are outside the remit of this review. The authors of this systematic review concluded that “regarding biomarkers of biological effect [another descriptor used in the literature for biomarkers of potential harm], the results show that levels found during the use of both e-cigarettes and heated tobacco products were generally moved in a direction believed to be consistent with improved health outcomes” and “since studies on biomarkers of biological effect may require longer intervention periods, the number of reports was limited without the necessary follow up time to show changes in biological functions” (Akiyama and Sherwood, 2021). The pooled analysis combined haematological data from three clinical studies in which measurements were taken three days after switching to use of a heated tobacco product or stopping smoking from conventional cigarette use (Roethig et al., 2010). Switching to use of a heated tobacco product or stopping smoking resulted in statistically significant decreases of up to 9% in haematological parameters including white blood cell count within three days. The second review detailed the quantitative results of a PMI study conducted in Japan and published in 2018 (Lüdicke et al., 2018b) as well as those of an unpublished study conducted in the USA and concluded that “studies conducted in people using Philip Morris International’s IQOS heated tobacco product did not reveal detectably better measures of biomarkers of potential harm than conventional cigarettes in human test” (Glantz, 2018b).\n\nTo date, two study protocols have been published that describe clinical trials currently underway, which include some component related to heated tobacco products and biomarkers of exposure and/or potential harm.\n\nThe first study protocol described a prospective study that aims to compare changes in cigarette consumption and adoption rates among smokers randomised to use of either heated tobacco products or vaping products (Caponnetto et al., 2020). As part of the study design, 220 healthy smokers, who are not motivated to quit, will be randomised into a 12-week single-centre, open-label, study where the primary outcome will be biochemically-verified self-reported continuous smoking abstinence at 12 weeks from the previous visit. Secondary outcomes will include levels of selected biomarkers of exposure in exhaled breath (exhaled carbon monoxide) and in spot urine samples (total NNAL, HEMA, MHBMA, HMPMA, 3-HPMA, S-PMA, AAMA, GAMA, CEMA, 2-HPMA, 1-OHP and cotinine). The second study protocol described a controlled, single-centre study involving 60 healthy subjects, divided in 6 groups (5 nicotine product user groups and 1 non-user group) based on their sole use of the products of choice (Sibul et al., 2021). The subjects were confined in a clinical setting for a period of 76 hours during which unrestricted use of their product of choice was provided (conventional cigarettes, heated tobacco products, EVPs, oral tobacco products and oral/dermal nicotine replacement therapy products). The aim of the study is to identify biomarkers and/or biomarker patterns in body fluids which may be used to distinguish between product use categories as a means to the determination of product use compliance in long-term clinical studies.\n\nThe results discussed in this section of the review clearly demonstrate that use of heated tobacco products in both clinical and “real-world” settings is associated with a statistically significant reduction in levels of non-nicotine related biomarkers of exposure to select toxicants associated with cigarette smoking. With respect to biomarkers of potential harm, the results indicate no statistically significant differences for the majority of the markers. This observation may be due to the follow-up periods reported by the studies being insufficient in length to observe physiologically favourable changes. It should also be noted that whilst the biomarkers of potential harm investigated in these studies may reflect processes on the pathway to smoking-related disease, their predictive and discriminative power has yet to be established so further studies such as long-term epidemiological studies are needed to show their relevance to tobacco-related disease and the subsequent impact of transitioning to heated tobacco product use (Akiyama and Sherwood, 2021). Clinical studies investigating heated tobacco product use and associated biomarkers of exposure and potential harm are currently underway. The results from these studies will further add to the available scientific literature.\n\nFor those HPHCs for which suitable biomarkers of exposure are not currently available, a modelling approach described as “nicotine bridging” has been proposed (Urban et al., 2012). In this model, exposure to HPHCs is estimated by quantifying levels of the HPHC under investigation in mainstream smoke/aerosol of the product under investigation followed by in vitro toxicity parameter-to-nicotine regressions after use of several machine-smoking protocols. Exposure to the HPHC under investigation is then modelled using nicotine pharmacokinetic data from clinical trials. Using this approach and data from a separate study relating to two conventional cigarettes and a heated tobacco product (Zenzen et al., 2012), exposure to several HPHCs for which biomarkers of exposure are not currently available was reduced for the heated tobacco product compared to either of the conventional cigarettes (Urban et al., 2012).\n\n\nHealth effects\n\nThis section of the review will discuss those articles that have investigated the potential health effects associated with heated tobacco product use.\n\nAcute exposure studies (≤1 month)\n\nSeveral separate reviews and editorials have discussed the potential pulmonary and cardiovascular effects associated with heated tobacco product use (Conklin et al., 2019; Biondi Zoccai et al., 2020a; Fried and Gardner, 2020; Münzel et al., 2020; Peruzzi et al., 2020a; St Claire et al., 2020; Bravo-Gutiérrez et al., 2021). However, most have provided only minimal discussion of quantitative data relating specifically to heated tobacco product use in experimental volunteers (Biondi Zoccai et al., 2020a; Münzel et al.,2020; Peruzzi et al., 2020a) whilst others have referenced no studies at all relating specifically to the cardiovascular and/or pulmonary effects of heated tobacco product use in humans (Conklin et al., 2019; St Claire et al., 2020; Bravo-Gutiérrez et al., 2021). Only a single review has provided a detailed discussion of the current literature on the potential cardiovascular effects of heated tobacco product use (Fried and Gardner, 2020). The authors of this review concluded that “current evidence suggests that heat-not-burn tobacco products (and electronic cigarettes) are less dangerous than combustible cigarettes, but not without health risk” and that “further clinical, animal, and in vitro studies must be developed to explore the cardiovascular effects of heat-not-burn tobacco products” (Fried and Gardner, 2020).\n\nIn the first reported study, eighteen adult conventional cigarette smokers were randomised to continued use of conventional cigarettes, use of a second-generation heated tobacco product (Accord) or abstinence from any form of product use for a period of three days prior to symptom-limited spiroergometry, as an estimation of exercise performance, in a three-period crossover study (Unverdorben et al., 2007). Use of the heated tobacco product resulted in less severe shortness of breath and higher working capacity, peak oxygen uptake, anaerobic threshold and maximum rate-pressure when compared to continued smoking of conventional cigarettes. Subsequent articles which reported on the same experimental design indicated a reduction in heart rate and rate-pressure product with both use of the same heated tobacco product and smoking abstinence when compared to continued use of the conventional cigarettes (Unverdorben et al., 2008), as well as an increase in heart rate variability as determined through use of 24-hour electrocardiogram (ECG) measurements (Munjal et al., 2009) and a physiologically favourable increase in pulmonary function parameters (Unverdorben et al., 2010). It should be noted, however, that the observed effects in these studies were more pronounced with smoking abstinence than with use of the heated tobacco product when compared to continued smoking of conventional cigarettes.\n\nWith respect to the potential cardiovascular and respiratory effects of those heated tobacco products which are currently commercially available, several experimental clinical studies have been reported.\n\nThe first study assessed the acute effects of smoking a single conventional cigarette (Marlboro Gold; nicotine content of 0.60mg per cigarette), taking nine puffs on an e-vapour product (blu Pro; 0.58mg nicotine content in 9 puffs based on a cartridge with a nicotine content of 16mg for 250 puffs), or consumption of a single heated tobacco product (IQOS; nicotine content of 0.50mg per stick) on several cardiovascular end-points including oxidative stress, antioxidant reserve, platelet activation and endothelial dysfunction (Biondi-Zoccai et al., 2019). Twenty healthy conventional cigarette smokers were assigned to use each product in a randomised order with a one-week washout period between each product use. Blood samples were taken just before and immediately after use of each allocated product and analysed for markers of oxidative stress (as determined by levels of sNox2-dp, a small peptide released after platelet activation, which is a measure of Nox2 activation, H2O2 production and 8-iso-PGF2α), antioxidant reserve (as determined by levels of vitamin E and HBA [serum H2O2 breakdown activity]), platelet function (as determined by levels of sCD40L and soluble P-selectin, two markers of platelet activation) and endothelial dysfunction (as determined by flow-mediated dilation, NO availability and blood pressure). Single use of any of the product was reported to have an adverse impact on the assessed cardiovascular parameters. However, heated tobacco product use was reported to have a less significant effect than e-vapour and conventional cigarette use on sNox2-dp levels (p=0.004 and p=0.001 respectively), 8-iso-PGF2α levels (p=0.004 and p<0.001 respectively) and vitamin E levels (p=0.018 and p=0.044 respectively). Heated tobacco product and e-vapour use were reported to have a comparably lesser effect than conventional cigarette use on flow-mediated dilation (p=0.872 for heated tobacco product versus e-vapour use), H2O2 production (p=0.522 for heated tobacco product versus e-vapour use), HBA (p=0.091 for heated tobacco product versus e-vapour use), sCD40L (p=0.849 for heated tobacco product versus e-vapour use) and soluble P-selectin sCD40L (p=0.821 for heated tobacco product versus e-vapour use). A subsequent study from the same researchers suggested that individual responses to acute heated tobacco product use differed specifically with respect to oxidative stress and platelet aggregation based on cluster analysis of data derived from 60 individuals including those who participated in their first study (Frati et al., 2020).\n\nTwo studies have reported the effects of acute heated tobacco product on experimental parameters of arterial stiffness (Ioakeimidis et al., 2020; Franzen et al., 2020). In the first study, 22 current conventional cigarette smokers were randomly assigned to use of either a heated tobacco product (IQOS), a conventional cigarette or a sham cigarette for a five-minute period during three separate experimental sessions which were conducted at least two days apart from each other (Ioakeimidis et al., 2020). The mean nicotine content for both the heated tobacco product and conventional cigarette was reported to be 0.5mg. Each experimental session was conducted in the morning after a minimum four hour fasting period during which time the subjects did not smoke or consumer any caffeinated drinks. Heart rate, blood pressure (both brachial and aortic), augmentation index corrected for heart rate (AIx@75), carotid-femoral pulse wave velocity (cfPWV) and brachial-ankle pulse wave velocity (bcPWV) were assessed immediately before and after smoking and then 5, 10, 20 and 30 minutes after product use. Baseline measurements were comparable across all three sessions with no statistically significant differences. Heart rate increased in a similar fashion after both conventional cigarette and heated tobacco product use (with a maximum increase of 10 beats per minute). Both brachial and aortic systolic blood pressure increased immediately after the end of smoking of conventional cigarettes (by 11.5 and 10.5mmHg; p<0.001 and p<0.01 respectively) and the heated tobacco product (by 7.5 and 6mmHg; p<0.01 in both cases). Blood pressure responses from baseline between the two product groups were not statistically significant at any time point through the experimental sessions (p>0.05). Compared with sham smoking, cfPWV, bcPWV and AIx@75 increased immediately after the end of conventional cigarette use (by 0.29m/s, 93cm/s and 3.3% respectively) as well as after heated tobacco product use (by 0.30m/s, 86cm/s and 3.5% respectively). Whilst heated tobacco product use when compared with conventional cigarette use resulted in less potent numerical increases in arterial stiffness indices after the end of smoking, the changes between the two product types were not different. The mean differences of cfPWV, bcPWV and AIx@75 area-under-the-curves between heated tobacco product use and conventional cigarette use (by 0.06m/s, 4.50cm/s and 1.97% respectively) were all statistically insignificant (all p>0.05). The authors concluded that “in the present cross-over, randomized trial comparing the acute effects of heat-not-burn cigarettes and tobacco cigarettes based on equivalent nicotine consumption in young smokers we found that use of any of these two products was associated with comparable acute detrimental effects on arterial stiffness” and that “it is likely that the acute effect of heat-not-burn cigarettes on arterial stiffness is mediated, at least in part, by nicotine and its effect on blood pressure”. The second study used a highly comparable experimental design with 20 healthy smokers and a two-hour follow-up after either smoking a conventional cigarette or use of the IQOS heated tobacco product (Franzen et al., 2020). Peripheral systolic blood pressure and heart rate increased significantly by more than 3% and 9% respectively compared to baseline after conventional cigarette and heated tobacco product use and returned to baseline levels after 60 and 45 minutes respectively. The augmentation index was significantly increased after 5, 10 and 15 minutes after conventional cigarette use and after 5 minutes for heated tobacco product use. The pulse wave velocity showed a trend towards being altered but this did not meet statistical significance (p=0.066).\n\nWith respect to pulmonary function parameters, the acute effect of heated tobacco product use in both smokers and non-smokers has been investigated (Pataka et al., 2020; Polosa et al., 2021a). In the first study, a total of 50 healthy male volunteers were recruited to the study (25 non-smokers and 25 current conventional cigarette smokers) (Pataka et al., 2020). Subjects underwent exhaled CO measurement, pulse oximetry (to determine blood oxygen saturation levels), pulmonary function tests (forced expiratory flow at 25% and 50% of vital capacity; FEF25% and FEF50%), peak expiratory flow (PEF) and airway resistances before and immediately after use of an IQOS heated tobacco product over a total of five to six minutes. Overall, oxygen saturation levels (98.4±1.2% before and 97.9±1.06% after; p=0.002), FEF25% (7.38±1.9L before and 6.98±1.92L after; p=0.002), FEF50% (5.00±1.42L before and 4.84±1.45L after; p=0.03) and PEF (7.9±2.16L before and 7.3±2.08L after; p<0.001) decreased significantly after use of the IQOS heated tobacco product whilst exhaled CO (1.75±1.02ppm before and 4.89±1.4ppm after respectively; p<0.001) and airway resistances at all tested frequencies (5, 10, 20, 25 and 35Hz) increased significantly. The authors concluded that “IQOS had an impact on exhaled CO, blood oxygen saturation and airway function immediately after use. Even those these changes were rather small to be considered of major clinical importance, they should raise concerns regarding the long-term safety of this product”. In the second study, the effect of heated tobacco product use on mucociliary clearance was investigated using saccharin test transit time (Polosa et al., 2021a). The saccharin test transit time was assessed in 39 current conventional cigarette smokers, 40 former conventional cigarette smokers, 40 never smokers and in 20 exclusive e-vapour users and 20 exclusive heated tobacco product users. The exclusive heated tobacco product users had not smoked conventional cigarettes for at least three to six months after switching to their heated tobacco product, had an exhaled carbon monoxide level of less than 7ppm and had been using heated tobacco products for a median of seven months. Conventional cigarette smokers had a median saccharin test transit time of 13.15 minutes which was significantly longer compared with that of all other groups. Exclusive heated tobacco product users had a median saccharin test transit time of 8.00 minutes which was not statistically different from that of exclusive e-vapour users (7.00 minutes), former conventional cigarette smokers (7.26 minutes) and never smokers (7.24 minutes).\n\nUsing transthoracic echocardiography, the acute effects of heated tobacco product use on myocardial systolic and diastolic function has been investigated (Yaman et al., 2021), involving thirty-eight current IQOS users. Volunteers used their own IQOS product which were all adjusted to achieve a two-second puff duration. Transthoracic echocardiography was performed three times for each volunteer: prior to use of any tobacco product, ten minutes after use of the IQOS heated tobacco product and ten minutes after smoking a single conventional cigarette. Each volunteer was randomised with respect to product use order with measurements being conducted on separate days. A total of ten puffs were taken for each product over a five-minute period. Heart rate increased significantly after heated tobacco product use (74.4±9.4bpm before and 81.8±8.7bpm after; p<0.01) whilst systolic blood pressure (111.3±13.5mmHg before and 114.1±16.8mmHg after; p=0.229) and diastolic blood pressure (71±10mmHg before and 71.9±10.1mmHg after; p=0.515) did not vary. All three parameters increased to a statistically significant extent after smoking a single conventional cigarette. With respect to transthoracic echocardiography parameters, heated tobacco product and conventional cigarette use resulted in a decrease in left ventricle global longitudinal strain, left ventricle global circumference strain and right ventricle global longitudinal strain compared to baseline values. No indication was provided as to whether or not these parameters returned to baseline vales and, if so, after what period of time.\n\nThe most recent study investigated the effect of heated tobacco product use (IQOS) and conventional cigarette use (Marlboro Red) on myocardial, coronary and arterial function in addition to oxidative stress and arterial function in 75 current conventional cigarette smokers using both acute (60-minute) and chronic (1-month) exposure scenarios (Ikonomidis et al., 2021). In the acute exposure scenario, 50 current conventional cigarette smokers were randomised to use of a single conventional cigarette or use of a single heated tobacco product followed with being crossed over to the alternative product after one hour. In the chronic exposure scenario, 50 current conventional cigarette smokers were switched to use of the heated tobacco product and compared to a separate group of 25 conventional cigarette smokers before and after one month of product use. Exhaled carbon monoxide, pulse wave velocity, malondialdehyde and 11-dehydrothromboxane B2 were assessed in both exposure scenarios whilst global longitudinal strain, myocardial work index, wasted myocardial work, coronary flow reserve, total arterial compliance and flow-mediated dilation were assessed in the chronic exposure scenario only. Acute use of the heated tobacco product resulted in a smaller increase in pulse wave velocity when compared to use of a conventional cigarette (change of 1.1m/s and 0.54m/s respectively; p<0.05). No statistically significant changes in exhaled carbon monoxide levels were reported after acute heated tobacco product compared to baseline values (14.2±7.3ppm and 14.9±7.4ppm respectively; p=0.1) whilst levels were significantly elevated following conventional cigarette use compared to baseline values (17.5±7.8ppm; p<0.001). Levels of malondialdehyde and 11-dehydrothromboxane B2 followed a comparable pattern with levels increased after conventional cigarette use, but not after heated tobacco product use, when compared to baseline levels. Malondialdehyde levels at baseline, after heated tobacco product use and after conventional cigarette use were 1.34±0.72nmol/L, 1.28±0.95nmol/L (p=0.55) and 2.56±0.85nmol/L (p=0.03) respectively. 11-dehydrothromboxane B2 levels at baseline, after heated tobacco product use and after conventional cigarette use were 378±103pg/ml, 362±113pg/ml (p=0.16) and 398±103pg/ml (p=0.02) respectively. With respect to the chronic exposure scenario, switching to the heated tobacco product resulted in statistically significant improvements in exhaled carbon monoxide levels, flow-mediated dilation, coronary flow reserve, total arterial compliance, global longitudinal strain, wasted myocardial work, malondialdehyde and 11-dehydrothromboxane B2 with differences of 10.42ppm, 4.3%, 0.98, 1.8ml/mmHg, 2.35%, 19.72mmHg%, 0.38nmol/L and 45pg/ml respectively (p<0.05 in each instance).\n\nChronic use studies (>1 month)\n\nTwo studies have reported on the potential cardiorespiratory effects of heated tobacco product use over a time-period exceeding one month.\n\nIn the first study, markers for oxidative stress, endothelial dysfunction and platelet activation were quantified in twenty chronic heated tobacco product users, twenty chronic conventional cigarette smokers and twenty non-smokers (Loffredo et al., 2021). Chronic use was defined as use exceeding a one-month period. All heated tobacco product users were reported as being former conventional cigarette smokers and had used their heated tobacco product for a mean of eighteen months. Oxidative stress was assessed by quantification of sNox2-dp levels and H2O2 production, platelet activation was assessed by quantification of platelet aggregation, sCD40L and soluble P-selectin levels and endothelial dysfunction was assessed by quantification of flow-mediated dilation and NO availability. Both measures of oxidative stress were increased in conventional cigarette smokers and heated tobacco product users compared to non-smokers with sNox2-dp showing statistically elevated levels in conventional cigarette smokers compared to heated tobacco product users whilst H2O2 production showed no statistically significant difference between use of the two product types. A comparable relationship was observed for endothelial dysfunction with conventional cigarette use having a more profound effect on flow-mediated dilation than the heated tobacco product whilst both product types produced comparable decreases in NO availability. With respect to platelet activation, heated tobacco product use produced less profound increases in sCD40L and soluble P-selectin compared with conventional cigarette use although differences in sCD40L levels were not statistically significant whilst differences in soluble P-selection levels were.\n\nIn the second study, health parameters were monitored for a period of three years in Chronic Obstructive Pulmonary Disease (COPD) smoking patients who substantially attenuated or ceased conventional cigarette consumption after transitioning to heated tobacco products (Polosa et al., 2021b). Changes in daily cigarette consumption, annualised disease exacerbations, lung function indices, self-reported outcomes and 6-minute walk distance from baseline were measured in the COPD patients using heated tobacco products after 12, 24 and 36 months. These were compared to a group of age and gender-matched COPD patients who continued to smoke conventional cigarettes throughout the 3-year period. Data was obtained for a total of 38 patients (with 19 in each group). COPD patients who transitioned to use of a heated tobacco product reported a substantial decrease in annualised COPD exacerbations from 2.1±1.9 (mean±SD) at baseline to 1.4±0.8, 1.2±0.8 and 1.3±0.8 at 12, 24 and 36-month follow up (p<0.05 for all time-points). No statistically significant changes were observed in COPD patients who continued to smoke conventional cigarettes over the same time period.\n\nA single study has reported on the incidence of acute exposure to heated tobacco products as documented by a national poison control centre.\n\nThe study reported a statistical analysis of calls received by the Czech Toxicological Information Centre over a seven-year period from 2012 to 2018 (Obertova et al., 2020). A total of 148 calls were received over the seven-year period in relation to exposures to vaping products, e-liquids or heated tobacco products (with three of these calls relating to animal exposures rather than human exposures). This cohort of 148 calls was equivalent to 0.12% of all calls received during the seven-year period (n=119,229). Heated tobacco products were reported as being the source of the acute exposure in 9 of the 148 calls (6%). The authors described exposure as being exposed to the “heat-not-burn cigarette refill” and these were reported by the authors as containing 5mg of nicotine. No further information was provided in the article with respect to heated tobacco products, as the authors did not stratify the results of their statistical analyses with respect to EVPs and heated tobacco products, but rather classed them together as a single product category.\n\nTo date, five medical case reports have been published in the scientific literature pertaining to heated tobacco products (Kamada et al., 2016; Aokage et al., 2019; Hitosugi et al., 2019; Tajiri et al., 2020; Yumoto et al., 2020). All of the medical case reports originated in Japan; three involved the development of acute eosinophilic pneumonia in users of heated tobacco products after normal use of their products (Kamada et al., 2016; Aokage et al., 2019; Tajiri et al., 2020) whilst two involved the intentional misuse of heated tobacco products or their constituents (Hitosugi et al., 2019; Yumoto et al., 2020). Of these two medical case reports, one detailed a case of attempted homicide in a user after tampering of his heated tobacco product with elemental mercury by a third party (Hitosugi et al., 2019), whilst the second involved the intentional ingestion of sticks associated with a heated tobacco product (Yumoto et al., 2020). Of the five medical case reports, two provided details as to the heated tobacco product involved (Hitosugi et al., 2019; Yumoto et al., 2020), two provided no details (Aokage et al., 2019; Tajiri et al., 2020) whilst for the final medical case report, the heated tobacco product involved could be deduced from one of the pictures provided in the original article (Kamada et al., 2016). An overview of the five medical case reports is provided in Table 5.\n\nF, female; M, male.\n\n1 Normal use defined as use of the heated tobacco product as per the manufacturer’s instructions. Intentional misuse defined as use of a heated tobacco product, or any of its constituent parts, as part of a deliberate attempt to cause harm to either yourself or others.\n\n2 Presumed to be IQOS based on the picture shown in Figure 1 of the original article.\n\n3 Both of these patients were reported by a separate author as not being current conventional cigarette smokers (Chaaban, 2020). However, no such information was identified by this author in either of the two original medical case reports.\n\n4 The patient had a significant known history of conventional cigarette use (27 years) and had switched to heated tobacco products from conventional cigarettes. In the two other patients diagnosed with acute eosinophilic pneumonia detailed above (Kamada et al., 2016; Aokage et al., 2019), it is not known whether or not they had previously smoked, or were current smokers, of conventional cigarettes.\n\nIt should be noted that those medical case reports which described health effects resulting from normal use of heated tobacco products (Kamada et al., 2016; Aokage et al., 2019; Tajiri et al., 2020) can only provide anecdotal evidence as it is possible that the observed health effect (reported as acute eosinophilic pneumonia in all three cases) could have been caused by an another factor or exposure not reported in the original articles or not reported by the patients to the authors of the original articles. Acute eosinophilic pneumonia is a rare disorder characterised by marked accumulation of eosinophils in lung tissues and/or bronchoalveolar fluid with most patients recovering completely following treatment with corticosteroids (Suzuki and Suda, 2019). It is of note that tobacco product use has been separately reported to be a significant factor in the development of acute eosinophilic pneumonia (Chaaban, 2020; Sakao, 2020).\n\nTo date, four epidemiological studies have been reported which have attempted to correlate heated tobacco product use with health-related endpoints.\n\nUsing cross-sectional data derived from a cohort of 58,336 Korean adolescents aged between twelve and eighteen years of age, one study attempted to assess the association between heated tobacco product use and the risk of allergic diseases (Lee et al., 2019). The data was derived from the 2018 Korea Youth Risk Behaviour Survey, and of all participants included in the survey, 2.4% (n=1,443), 20.9% (n=11,884) and 7.2% (n=4,198) reported a diagnosis of asthma, allergic rhinitis and atopic dermatitis within the previous year respectively. Conventional cigarettes, vapour products and heated tobacco products were reported as being used by 13.9% (n=8,129), 7.1% (n=4,114) and 2.4% (n=1,414) of participants, respectively. 82.4% of heated tobacco products were reported as being triple users (concurrent use of all three product types). Sole use of heated tobacco products was associated with a statistically significant increased risk of asthma; dual use of heated tobacco products and conventional cigarettes was associated with a statistically significant increased risk of asthma and atopic dermatitis whilst triple use of heated tobacco products, conventional cigarettes and vapour products was associated with a statistically significant increased risk of asthma, allergic rhinitis and atopic dermatitis. Dual use of heated tobacco products and vapour products was not associated with a statistically significant risk for any of the three allergic diseases. However, limitations such as the cross-sectional nature of the study and survey design prevents a causal relationship from being established.\n\nA subsequent article used data from the same survey with different inclusion criteria to assess the association between heated tobacco product use and the risk of allergic rhinitis and asthma (Chung et al., 2020). In this study, the researchers used a cohort of 60,040 Korean adolescents aged between thirteen and eighteen years of age. Within the cohort, 2.5% and 20.8% of participants reported having prevalent asthma and allergic rhinitis respectively. 2.9% of the participants (n=1,568) reported themselves as ever having used a heated tobacco product. With respect to allergic rhinitis, ever use of a heated tobacco product alone [combined with never use of both conventional cigarettes and vapour products] did not increase the risk of developing the condition (odds ratio of 1.9; 95% confidence intervals of 0.7 to 5.1). The sole product use scenarios which increased the risk for developing allergic rhinitis were ever use of heated tobacco products combined with both former use of vapour products and former use of conventional cigarettes (odds ratio of 1.9; 95% confidence intervals of 1.1 to 3.2) and ever use of heated tobacco products combined with current use of both vapour products and conventional cigarettes (odds ratio of 1.6; 95% confidence intervals of 1.1 to 2.2). With respect to asthma, ever use of a heated tobacco product alone [combined with never use of both conventional cigarettes and vapour products] increased the risk of developing the condition (odds ratio of 3.8; 95% confidence intervals of 1.5 to 9.6). The other product use scenarios which increased the risk for developing asthma were ever use of heated tobacco products combined with never use of vapour products and current use of conventional cigarettes (odds ratio of 6.0; 95% confidence intervals of 3.1 to 11.5) and ever use of heated tobacco products combined with current use of both vapour products and conventional cigarettes (odds ratio of 1.7; 95% confidence intervals of 1.1 to 2.7). It should be noted that the statistically significant observation that ever use of a heated tobacco product alone [combined with never use of vapour products and conventional cigarettes] resulted in an increase in risk of developing asthma was based on only fifty-nine individuals (out of 60,040 in the cohort) who reported ever use of a heated tobacco product and never use of both vapour products and conventional cigarettes.\n\nA subsequent study aimed to investigate the potential association between conventional cigarette use, heated tobacco product use and dual product use and self-reported periodontal disease using data from the 2019 arm of the Japan “Society and New Tobacco” internet survey (JASTIS) (Yoshioka and Tabuchi, 2021). Of the 10,439 JASTIS survey respondents, the number of current exclusive conventional cigarette smokers, current exclusive heated tobacco product users, and current dual product users was 1,304, 437 and 1,049 respectively. Compared with never users, current exclusive heated tobacco product use was significantly associated with an increased prevalence of self-reported periodontal diseases (prevalence ratio of 1.43; 95% CI of 1.08 to 1.88). Compared with never users, current exclusive conventional cigarette smoking (prevalence ratio of 1.29; 95% CI of 1.03 to 1.62) and current dual product use (prevalence ratio of 1.55; 95% CI of 1.20 to 1.99) were also significantly associated with an increased prevalence of self-reported periodontal diseases.\n\nThe most recent epidemiological study investigated the association between heated tobacco product use and respiratory symptoms in Hong Kong adolescents (Wang L et al., 2021). Using a cross-sectional school-based survey which provided an anonymous questionnaire to 33,627 students, the association between persistent respiratory symptoms and use of heated tobacco products was investigated. The main outcome of the study was self-reported respiratory symptoms which lasted for three consecutive months in the previous twelve months. Compared to never users of heated tobacco products, both former users (prevalence ratio of 1.30; 95% confidence intervals of 1.06 to 1.59) and current users (prevalence ratio of 1.59; 95% confidence intervals of 1.23 to 2.06) were more likely to self-report respiratory symptoms. Compared to current exclusive conventional cigarette smokers, both current exclusive heated tobacco product users (prevalence ratio of 1.40; 95% confidence intervals of 0.93 to 2.11) and current dual users of both products (prevalence ratio of 1.19; 95% confidence intervals of 0.94 to 1.49) were not more likely to self-report respiratory symptoms.\n\nIn addition to those studies discussed in this review, several study protocols have been published for clinical trials currently underway which are investigating potential health effects associated with heated tobacco products and which will likely produce articles of interest in the future.\n\nA study protocol for a five-year single centre observational study, partially funded by PMI, has been published which aims to evaluate frequency of exacerbations, respiratory symptoms, physical exercise intolerance and abnormal lung functions in men and women aged 40 to 59 years who live in Almaty, Kazakhstan who use the IQOS heated tobacco product compared to those who smoke conventional cigarettes (Sharman et al., 2018). Participant recruitment began in December 2018 and enrolment was expected to last until late summer 2018. The authors reported that the study results will be published in a peer-reviewed scientific journal once the study has been completed.\n\nPMI have completed a randomised, controlled two-arm parallel-group multicentre Japanese study which investigated the effect of switching to a heated tobacco product on periodontal endpoints in patients with generalised chronic periodontitis (Pouly et al., 2021). A total of 172 subjects were randomised to continued conventional cigarette smoking (n=86) or switching to the heated tobacco product with all subjects completing the study. The conduct phase of the study has been completed and the data cleaning and statistical analyses currently underway.\n\nThe SUR-VAPES 3 study aims to compare the acute coronary effects of EVP and heated tobacco product use in chronic conventional cigarette smokers admitted to hospital for invasive coronary evaluation (Biondi Zoccai et al., 2020b). In patients with a confirmed angiographic intermediate coronary stenosis, who have never quit smoking before, the study will measure coronary flow reserve as the primary end-point. Twenty patients will be randomised to either single use of an EVP (n=10) or single use of a heated tobacco product (IQOS; n=10) whilst in the catheterisation laboratory, followed by repeat coronary flow reserve measurement (Lombardi et al., 2020).\n\nThe DIASMOKE study aims to investigate whether or not switching from conventional cigarettes to NGPs (including EVPs or heated tobacco products) in type 2 diabetic smokers will yield a quantifiable improvement in metabolic syndrome factors within a two-year time period (Krysinski et al., 2021). The study aims to recruit a total of 576 diabetic patients who will be randomised in a non-blinded fashion to receive either a referral to smoking cessation services or to receive an NGP of their choice. It is anticipated that results will be available between 2023 and 2024.\n\nThe 12-month randomised controlled trial involving “real-world” use of heated tobacco products which is currently being conducted by BAT includes several health-related secondary endpoints in its study design (Newland et al., 2019; Camacho et al., 2020). These include augmentation index, pulse wave velocity, reactive hyperaemia index, lung spirometry and brachial systolic and diastolic blood pressure measurements with measurements throughout the 12-month period. Interim analyses up to 90 and 180 days have been published (Gale et al., 2021; Gale et al., 2021b) and it is anticipated that future publications relating to the 12-month analysis will include data relating to these secondary endpoints.\n\nThe results discussed in this section of the review demonstrate that whilst several studies have reported that the use of heated tobacco products under acute exposure settings may have both physiologically favourable and unfavourable effects on both the cardiovascular and pulmonary systems, studies into their long-term health effects are currently lacking. Of the two available studies into long-term health effects, one reported physiologically favourable outcomes (Polosa et al., 2021b) whilst the other did not (Loffredo et al., 2021). There are currently no studies on the potential risk of heated tobacco product use on development of any form of cancer and long-term epidemiological studies into this may be warranted.\n\nThere are suggestions from medical case reports that the use of heated tobacco products may be associated with the development of the rare disorder, acute eosinophilic pneumonia (Kamada et al., 2016; Aokage et al., 2019; Tajiri et al., 2020). However, the number of reported cases of acute eosinophilic pneumonia is extremely small compared to the number of heated tobacco product users. Furthermore, there is no data currently available to indicate whether or not the incidence of acute eosinophilic pneumonia secondary to use of heated tobacco products is higher than that associated with conventional cigarette use.\n\n\nHeated tobacco products, indoor air quality and bystander exposure\n\nThis section of the review will discuss those articles which have investigated the effects of heated tobacco product use on indoor air quality (IAQ) and the potential health effects of bystander exposure.\n\nThe International Agency for Research on Cancer has concluded that there is sufficient evidence in humans for the carcinogenicity of environmental tobacco smoke (ETS) produced through use of conventional cigarettes and other combustible tobacco products within indoor environments (International Agency for Research on Cancer, 2012). In addition, the United States Surgeon General has concluded that there is no risk-free level of exposure to ETS and that exposure to ETS in adults has immediate adverse effects on the cardiovascular system and causes coronary heart disease and lung cancer (United States Surgeon General, 2006).\n\nGiven these findings from the public health community, there is concern that use of heated tobacco products in indoor environments may negatively impact IAQ and may subsequently result in health effects in bystanders (Tabuchi et al., 2018; Imura and Tabuchi, 2021). A web-based survey conducted in Japan in early 2018 reported that 15.6% of current tobacco users used heated tobacco products within indoor public spaces whilst 72.0% of the same survey respondents reported using conventional cigarettes within indoor public spaces (Sutanto et al., 2019). For dual users, conventional cigarette use within public indoor spaces was reported as being more frequent (97.7%) than heated tobacco product use (76.0%).\n\nETS released into indoor environments from the smoking of conventional cigarettes is an aged and diluted mixture comprised of sidestream smoke released directly from the cigarette and aerosol exhaled by the smokers after their inhalation of mainstream smoke (Tricker et al., 2009). The emissions present in indoor environments as a result of heated tobacco product use are principally comprised of aged and diluted aerosol exhaled by users after inhalation on the heated tobacco product, since there is no sidestream smoke produced by heated tobacco products, although an exploratory analytical analysis indicated some emissions may be released both whilst heated tobacco products are turned on and whilst they are actively being used (O’Connell et al., 2015).\n\nTable 6 details those experimental studies which have characterised the emissions present in indoor environments after controlled use of heated tobacco products. These studies investigated a range of historical and currently commercially available heated tobacco products including Accord, IQOS and glo and employed a range of analytical methodologies with some being specifically validated for use with heated tobacco products (Mottier et al., 2016; Gómez Lueso et al., 2018). Most of these studies have reported on IAQ levels before, during and after heated tobacco product use within an experimental indoor environment, with a single study reporting on IAQ levels during and after experimentally controlled heated tobacco product use within a non-operational nightclub which featured an external smoking area (Kaunelienė et al., 2019). Two additional studies reported on heated tobacco product use within different models of passenger car (Schober et al., 2019; Savdie et al., 2020) whilst a single study detailed the effects of heated tobacco product use on outdoor air quality (Cammalleri et al., 2020).\n\nIf the quantified chemical constituent was determined to be at below the limit of quantification or limit of detection of the analytical method employed then this is indicated by the abbreviation ND and dark green shading. If the quantified chemical constituent was determined to be at a significantly lower level within the indoor environment following heated tobacco product use when compared to conventional cigarette use then this is indicated by a downwards arrow and light green shading). 3-EP, 3-ethenylpyridine; AA, Acetaldehyde; CO, Carbon monoxide; CO2, Carbon dioxide; FPM, Fluorescing particulate matter; ND, Not detected [experimental results below the limit of detection or limit of quantification of the analytical method employed]; NH3, Ammonia; NO, Nitrogen oxide; NOx, Nitrogen oxides; PMx, Particulate matter with a mean aerodynamic diameter below x μm; RSP, Respirable suspended particulates; SMP, Submicronic particles; Sol, Solanesol; Sol-PM, Solanesol-related particulate matter; TVOC, Total volatile organic compounds; UVPM, Ultraviolet absorbing particulate matter.\n\nThe results shown in Table 6 indicate whether the quantified chemical constituent was determined to be at or below the limit of quantification or limit of detection of the analytical method employed (as indicated by the abbreviation ND and dark green shading) or at a significantly lower level within the indoor environment following heated tobacco product use when compared to conventional cigarette use (as indicated by a downwards arrow and light green shading). The aim of this approach is to demonstrate that heated tobacco product use under controlled conditions resulted in a quantifiable, and statistically significant, decrease in levels of chemical constituents when compared to conventional cigarette use under the same experimental conditions. The results from these studies, as shown in Table 6, indicate that a range of IAQ chemical constituent markers are either present in the experimental indoor environments at markedly lower concentrations when compared to levels observed with conventional cigarette use within the same indoor environments or determined to be below the level of detection or limit of quantification of the analytical methods employed.\n\nSeveral of the studies have compared their analytical measurements of IAQ markers with heated tobacco product use with occupational exposure limits and/or air quality guidelines. The authors of a controlled study using the THP1.0 heated tobacco product observed that “environmental emissions from the THP1.0 would conform to published IAQ guidelines, such as those from the World Health Organisation and, for particles, would conform to target the World Health Organisation outdoor air annual mean limits of 10μg/m3 PM2.5” (Forster et al., 2018b). Comparable conclusions have been reported for the THS2.2 heated tobacco product including with respect to its nicotine emissions (Mitova et al., 2016; Prodanchuk et al., 2017).\n\nUsing data from their own analytical assessment study (Hirano et al., 2020), separate authors attempted to estimate the excess cancer risk in bystanders exposed to either ETS or exhaled heated tobacco product aerosol under usual indoor conditions (Hirano and Takei, 2020). Based on the assumption that nicotine inhalation is proportional to cancer potency, the lifetime cancer risk for heated tobacco product exhaled aerosol exposure was estimated to be 2.7x10-6 (below 1x10-5; 1 in 100,000) with this being three orders of magnitude lower than that estimated for exposure to ETS (8.3x10-3) using the same approach.\n\nThere are currently no clinical or epidemiological studies which have attempted to associate exposure to emissions produced from heated tobacco product use within indoor environments with any subsequent potential health effects in bystanders.\n\nHowever, two surveys conducted in Japan in 2017 and 2019 respectively have investigated the frequency of self-reported symptoms occurring from such an exposure scenario (Tabuchi et al., 2018; Imura and Tabuchi, 2021). In the first survey, a total of 12% of the survey respondents reported as having been exposed to emissions from heated tobacco product use in their vicinity (977 respondents of a total survey size of 8,240 respondents) (Tabuchi et al., 2018). Of this group, 37% experienced at least one symptom as a result of the exposure with the most common complaint being feeling generally ill (25%) followed by eye discomfort/pain (22%), a sore throat (21%) and any other [unspecified] injury or symptom (13%). Only 26% of current heated tobacco product users reported having experienced at least one symptom compared to 41% of former users of heated tobacco products and 49% of never users of any tobacco product. In the second survey, a total of 33% of the survey respondents reported as having been exposed to emissions from heated tobacco use in their vicinity (2,923 respondents of a total survey size of 8,784 respondents) (Imura and Tabuchi, 2021). Of this group, 40% experienced at least one symptom as a result of the exposure with most common compliant being nausea (32%), respiratory symptoms (29%), sore throat (23%), cough (23%), eye pain (19%), headache (18%), chest pain (12%), any other [unspecified] symptom (12%) and asthma attack (11%).\n\nIn addition to the potential for inhalation of emissions produced by heated tobacco product use present by bystanders, it has also been claimed there is a potential for exposure through residues present on indoor surfaces which deposit onto them through diffusion and sedimentation after heated tobacco product use. Such an exposure scenario is referred to as “third-hand” exposure.\n\nUsing a novel aerosol exposure chamber, surface staining of wallpaper and cotton samples after exposure to mainstream smoke from 3R4F Kentucky Reference cigarettes, aerosol from a heated tobacco product (THP 1.0) and e-vapour products has been reported (Dalrymple et al., 2020). The exposure chamber had an internal volume of 885cm3 with the wallpaper and cotton samples being exposed to 200, 400, 600, 800 or 1,000 puffs of undiluted conventional cigarette mainstream smoke or heated tobacco product aerosol produced using the Health Canada Intense regime. After delivery of 50 puffs to the chamber, a settling time of five minutes was included to allow for aerosol deposition by diffusion and sedimentation within the chamber. Following exposure to 200 puffs, wallpaper and cotton samples were removed from the exposure chamber and staining levels determined quantitatively using a spectrophotometer. Exposure to between 200 and 1,000 puffs from the 3R4F Kentucky Reference cigarette yielded a visible dose-response effect with respect to the wallpaper and cotton samples whilst exposure to aerosols from both the heated tobacco product and EVPs after the same number of puffs showed relatively little colour changes. The authors concluded that “the method developed demonstrated that cigarette smoke exposure significantly increased the level of wallpaper and cotton sample staining in a dose dependent manner, whereas glo, glo pro, glo sens [heated tobacco products] or iSwitch Maxx [an EVP] exposure resulted in significantly reduced levels of staining. This data suggests that PRRPs [potentially reduced risk products] may have additional social benefits for consumers and others”.\n\nThe results discussed in this section of the review demonstrate that a range of IAQ chemical constituent markers are present at markedly lower concentrations when compared to levels observed with conventional cigarette use within indoor environments; these are typically comparable to background levels of below IAQ standards. Furthermore, the intensity of indoor surface staining is markedly less with heated tobacco product use when compared to conventional cigarette use and the lifetime cancer risk for heated tobacco product exhaled aerosol exposure has been estimated to be three orders of magnitude lower than that estimated for exposure to ETS when using the same risk assessment modelling approach.\n\nThese conclusions are comparable to those reported by separate authors from both independent and heated tobacco product manufacturers. A review on the impacts of use of the THS heated tobacco product on IAQ concluded that “generally, the usage of THS has been associated with lower or comparable indoor air pollutant concentrations compared against other conventional indoor sources or environments, in most cases distinguishable above background” (Kaunelienė et al., 2018) whilst a systematic review of heated tobacco products concluded that “evidence on heat-not-burn secondhand emissions suggested that heat-not-burn [tobacco product use] exposes users and bystanders to substantially lower but measurable levels of particulate matter and HPHC” (Simonavicius et al., 2019) and an analytical study concluded that “heated tobacco products are a weaker indoor pollution source than conventional cigarettes, but their impacts are neither negligible nor yet fully understood” (Cancelada et al., 2019). A further commentary also noted that “there is a reduction in risk to bystanders where conventional [cigarette] smokers switch to heat-not-burn products” (Górski, 2019). These conclusions are comparable to those reached by Public Health England who concluded that “compared with cigarettes, heated tobacco products are likely to expose users and bystanders to lower levels of particulate matter and harmful and potentially harmful compounds (HPHC). The extent of the reduction found varies between studies” (Public Health England, 2018).\n\nFinally, it should be noted that no clinical or epidemiological studies have been reported which have attempted to associate exposure to emissions produced from heated tobacco product use within indoor environments with subsequent health effects in bystanders. Such studies may be informative.\n\n\nOverall summary\n\nThe vast majority of the articles discussed in this review which provide quantitative data have concluded that heated tobacco products show a favourable tobacco harm reduction potential compared with conventional cigarettes. Although not risk free, this reduced risk profile may have been reported as a substantial decrease in levels of toxicants present in aerosol, coupled with lower levels of biomarkers of exposure, effect and/or function after controlled product use in clinical settings, with significantly lower biological effects in in vitro or in vivo experimental studies or lower levels of indoor air quality markers in indoor environments after controlled product use.\n\nWith respect to those studies published to date which have suggested that, overall, heated tobacco products may be equally harmful or more harmful than conventional cigarettes (Auer et al., 2017a; Chun et al., 2018; Leigh et al., 2018a; Nabavizadeh et al., 2018; Salman et al., 2019; Sohal et al., 2019; Ioakeimidis et al., 2020), the experimental findings can be either explained by reference to the known effects of nicotine where heated tobacco products deliver comparable levels of nicotine to conventional cigarettes (Nabavizadeh et al., 2018; Ioakeimidis et al., 2020), to the findings of other relevant studies which contradict their findings (Chun et al., 2018; Leigh et al., 2018a; Sohal et al., 2019), by limitations with respect to the experimental methodologies employed (Auer et al., 2017a) or due to expected differences between the compositions of tobacco rods between heated tobacco products and conventional cigarettes (Salman et al., 2019).\n\nTo date, no novel health effects have been associated with heated tobacco product use which have not been reported with the use of tobacco and/or nicotine containing products. Long term research is warranted in this regard. Three medical case reports of acute eosinophilic pneumonia have been published which reported that heated tobacco product use was associated with development of the condition (Kamada et al., 2016; Aokage et al., 2019; Tajiri et al., 2020) and the use of conventional cigarettes is known to be a significant risk factor for development of the condition (Chaaban, 2020). However, the number of reported cases of acute eosinophilic pneumonia is extremely small compared to the number of heated tobacco product users. Furthermore, there is no data currently available to indicate whether or not the incidence of acute eosinophilic pneumonia secondary to use of heated tobacco products is higher than that associated with conventional cigarette use.\n\nGiven the enormous tobacco harm reduction potential that heated tobacco products offer to adult smokers who would otherwise continue to smoke due to the substantial reduction in numbers and levels of toxicants present in their aerosols compared to combustible tobacco smoke, there is a need for more research that is independent of commercial interests to be conducted with data from independent sources required to validate industry findings (Stepanov and Woodward, 2018). The totality of the currently available scientific evidence indicates heated tobacco products are likely to be significantly less harmful than conventional cigarettes, and have a risk profile much closer to that of non-tobacco containing products (Leigh et al., 2018a; Stephens, 2018; McEwan et al., 2021). These findings are consistent with the concept of a continuum of risk for non-combustible tobacco and tobacco-free nicotine products (McAdam et al., 2018), and are supported by the conclusions from a recent UK government review on vapour products and heated tobacco products (Public Health England, 2018).\n\nGiven the limited time during which heated tobacco products have been commercially available, there is limited data on long term use and more research is needed. Such studies will be of significant benefit and there are indications that such studies are underway (Sharman et al., 2018; Newland et al., 2019; Camacho et al., 2020).\n\nFinally, the available scientific evidence suggests a significant reduction in smoking-related risk for conventional cigarette adult smokers who transition to heated tobacco products completely whilst quitting the use of all tobacco (and nicotine) products irrespective of their method of operation will lead to the greatest overall reduction in risk. This is consistent with public health messaging and the conclusions of separate authors (Slob et al., 2020).\n\n\nFuture research\n\nBased on the content of this review, there are several areas of research related to the potential health effects associated with heated tobacco products which may warrant further investigation:\n\n1. There is currently insufficient epidemiological data on the health effects of heated tobacco product use. To date, only four epidemiological studies have been published (Lee et al., 2019; Chung et al., 2020; Yoshioka and Tabuchi, 2021; Wang L et al., 2021). Further studies are warranted into the long-term health effects of heated tobacco product use.\n\n2. There is currently no epidemiological data in relation to the potential carcinogenicity of heated tobacco product use. Long-term epidemiological studies are warranted in this matter to determine if a transition from conventional cigarette use to heated tobacco product is associated with a decreased incidence of smoking-related cancers.\n\n3. There is currently a lack of clinical or epidemiological data on the effects of exposure to emissions produced from heated tobacco product use within indoor environments on potential health effects in bystanders. Acute exposure and long-term epidemiological studies would be informative.\n\n4. There is a currently insufficient experimental data in relation to the potential health effects associated with use of heated tobacco products in pregnancy. To date, no human data has been reported on the potential effects of heated tobacco product use in pregnancy on maternal and/or foetal health (Li et al., 2018; Kopa and Pawliczak, 2020). Currently, only a single in vivo animal study is available with investigates fetal effects of maternal exposure to heated tobacco products in mice (Yoshida et al., 2020). Although pregnant women should not use any tobacco or nicotine products, the potential risks to both mother and child associated with this exposure scenario should be investigated further.\n\n5. There is currently insufficient experimental data on the role of electrically heated tobacco product components on HPHC production. Whilst the overwhelming evidence indicates that aerosols experimentally produced from heated tobacco products contain lower quantities of HPHCs than mainstream smoke from both reference conventional cigarettes and commercially available conventional cigarettes, there is limited evidence to suggest that non-tobacco components of the heated tobacco products such as the filter present in the tobacco stick and the heating elements of the electrical device can produce chemical constituents of toxicological concern such as acrolein, cyanohydrin and formaldehyde in their own right (Davis et al., 2019b; Kim et al., 2020; Kim and An, 2020). Further studies are required to investigate this phenomenon.\n\n\nData availability\n\nOpen Science Framework: The product science of electrically heated tobacco products, https://doi.org/10.17605/OSF.IO/XQ3HB (Mason, 2021).\n\nThis project contains the following underlying data:\n\n- Supplementary_File_1.xlsx (Citation details for all journal articles identified in the review)\n\n- Supplementary_File_2.docx (Table detailing the studies available in the scientific literature providing quantitative experimental data on levels of HPHCs produced by heated tobacco products in direct comparison with those produced by conventional cigarettes)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAdamson J, Jaunky T, Thorne D, et al.: Characterisation of the borgwaldt LM4E system for in vitro exposures to undiluted aerosols from next generation tobacco and nicotine products (NGPs). 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PubMed Abstract \| Publisher Full Text\n\nCaponnetto P, Caruso M, Maglia M, et al.: Non-inferiority trial comparing cigarette consumption, adoption rates, acceptability, tolerability, and tobacco harm reduction potential in smokers switching to Heated Tobacco Products or electronic cigarettes: Study protocols for a randomized controlled trial. Contemp Clin Trials Commun. 2020; 17: 100518. Publisher Full Text\n\nCaputi TL, Leas E, Dredze M, et al.: They’re heating up: Internet search query trends reveal significant public interest in heat-not-burn tobacco products. PLoS One. 2017; 12: e0185735. PubMed Abstract \| Publisher Full Text\n\nCaruso M, Polosa R: Perplexing conclusions concerning heat-not-burn tobacco cigarettes. JAMA Intern Med. 2017; 177: 1699–1700. PubMed Abstract \| Publisher Full Text\n\nCaruso M, Emma R, Rust S, et al.: Screening of different cytotoxicity methods for the assessment of ENDS toxicity relative to tobacco cigarettes. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "135110", "date": "04 Jul 2022", "name": "Gagandeep Kaur", "expertise": [ "Reviewer Expertise Inhalation toxicology", "epigenetics", "smoking", "senescence" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current review is a very comprehensive review of the toxicological and risk assessment of electronically heated tobacco products. The authors have done a commendable job in putting together all the existing literature comparing the toxicology and health effects of aHTPs with conventional cigarettes. However this reviewer feels that the length of this review with so many different aspects being discussed makes it a tough read.\nHere are a few suggestions to improve upon this manuscript:\nThe way the review is structured it might as well be structured as a systematic review. Authors must check the guidelines and restructure the manuscript accordingly.\n\nThe manuscript will benefit much with some serious editing. Repetitions and redundant statements and studies can be removed from discussion and key ideas should be focused on in terms of the public health risk.\n\nThe quality of Tables could be improved.\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": "157707", "date": "21 Dec 2022", "name": "Jinsong Chen", "expertise": [ "Reviewer Expertise Tobacco control", "smoking cessation", "clinical trials" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a thorough, comprehensive account of the current scientific evidence on heated tobacco products and their potential health effects.\nThe authors have used a broad 'scoping review' approach to identifying key papers, studies and reviews pertinent to the topic. They used appropriate search terms and specifications. They provide a suitable flow chart to show the selection process they used to identify relevant articles. It is unlikely that their review has missed important reviews or studies.\nThey make a fair case in support of the overall evidence pointing to these products being less likely to cause harm than conventional combusted tobacco products because of the constituents in their emissions, their toxicological profiles from in vitro studies and in vivo animal studies, changes in biomarkers among users relative to smokers and non-users/non-smokers, and second-hand exposures. They identify several key issues that are 'live' and need resolution\nthere is a paucity of non-industry studies. Most research studies are tobacco industry-funded (including the present paper).\n\nThe conclusions tend to differ regarding assessment of harm, between industry and independent research.\nThe conclusions about where research in this field needs to go are correct: 1) first and foremost, far more independent (non-industry) research is needed; 2) there is diversity in assessment methods, especially between industry and independent researchers; and agreement is needed in the scientific community about the optimal standards for assessing harms; 3) a gap exists in evidence from long-term follow-up studies of the health of cohorts of heated product users compared with smokers and non-user/non-smokers; and another gap exists in research around population-level impacts of these products. Perhaps the best source of such data could come from Japan where heated tobacco products have emerged as a popular cigarette substitute. 4) Dual use (smoking conventional and heated products) needs further investigation. I would add to this list the combination of e-cigarette use and heated tobacco product use as a potential 'dual use' phenomenon that is likely to grow in popularity as changes continue to occur in the nicotine product marketplace.\nOverall, the paper is a comprehensive, balanced overview and makes a useful contribution to the literature on these novel products.\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/11-121
https://f1000research.com/articles/11-120/v1	31 Jan 22	{ "type": "Research Article", "title": "Quantification of the effects of chimerism on read mapping, differential expression and annotation following short-read de novo assembly.", "authors": [ "Raquel Linheiro", "John Archer", "Raquel Linheiro" ], "abstract": "Background: De novo assembly is often required for analysing short-read RNA sequencing data. An under-characterized aspect of the contigs produced is chimerism, the extent to which affects mapping, differential expression analysis and annotation. Despite long-read sequencing negating this issue, short-reads remain in use through on-going research and archived datasets created during the last two decades. Consequently, there is still a need to quantify chimerism and its effects.\n\nMethods: Effects on mapping were quantified by simulating reads off the Drosophila melanogaster cDNA library and mapping these to related reference sets containing increasing levels of chimerism. Next, ten read datasets were simulated and divided into two conditions where, within one, reads representing 1000 randomly selected transcripts were over-represented across replicates. Differential expression analysis was performed iteratively with increasing chimerism within the reference set. Finally, an expectation of r-squared values describing the relationship between alignment and transcript lengths for matches involving cDNA library transcripts and those within sets containing incrementing chimerism was created. Similar values calculated for contigs produced by three graph-based assemblers, relative to the cDNA library from which input reads were simulated, or sequenced (relative to the species represented), were compared.\n\nResults: At 5% and 95% chimerism within reference sets, 100% and 77% of reads still mapped, making mapping success a poor indicator of chimerism. At 5% chimerism, of the 1000 transcripts selected for over-representation, 953 were identified during differential expression analysis; at 10% 936 were identified, while at 95% it was 510. This indicates that despite mapping success, per-transcript counts are unpredictably altered. R-squared values obtained for the three assemblers suggest that between 5-15% of contigs are chimeric.\n\nConclusions: Although not evident based on mapping, chimerism had a significant impact on differential expression analysis and megablast identification. This will have consequences for past and present experiments involving short-reads.", "keywords": [ "Chimerism", "de novo assembly", "differential expression", "read mapping", "contig annotation", "Drosophila melanogaster" ], "content": "Introduction\n\nIn the absence of a closely related reference set of transcripts that describes what may be expressed within a transcriptome, de novo assembly can be a pivotal point for transcriptomic experiments utilizing short-read RNA sequencing (RNA-Seq) data.1–3 The general goal is to increase understanding of how cells, tissues and organs develop,4,5 adapt,6,7 function8,9 and interact10,11 within their respective environments under varying conditions. This can be achieved through the characterization of expressed genes,1,12 the identification of differentially expressed genes13 and genomic level annotation using expressed transcripts as a guide,14–16 along with other types of RNA-Seq data analysis.17 In experiments involving short-read RNA-seq data, de novo assembly refers to the construction of a set of contigs from the short-read data that can be used as a reference set, often for the characterization of transcript expression profiles.18,19 Transcriptomics experiments have an impact across the entire living world, including host-pathogen interactions,20,21 the development of diseases such as cancers,22 diabetes,23 heart disease24 and Alzheimer’s,25 diseases associated with ageing,26 as well as animal27,28 and plant29,30 domestication; the latter requiring persistent alterations selected for through many generations. From an early stage, much effort has been invested in the optimization of experimental design and data analysis strategies.31 For example, a source of error that arises involves base calling during the sequencing process,32,33 and there are approaches available for read trimming34 and error correction35–37 that combine to form effective solutions.\n\nA less obvious and less explored source of error is the erroneous chimeric sequences that can be introduced during the de novo assembly process.38–42 These are distinct from those introduced during library preparation.38,39 Non-chimeric contigs are assembled sequences that accurately represent expressed transcripts. Erroneous chimeric contigs occur when two or more fragments of DNA are incorrectly joined together. Primarily, the possibility of chimeric contigs arises when assembling portions of the read data representing increased biological complexity,40 such as transcripts expressed from multi-gene families.41,42 Many of the fundamental approaches to short-read assembly, including graph, reference and the overlap based methods, were developed in the early days of RNA-Seq read data analysis.43,44 The range of approaches developed, as well as the various parameter options explored,45–47 indicate the importance of this process. Combined with the variable results achieved,48–50 it is evident that a consensus “best approach” has not been resolved. More recent developments that incorporate the usage of long reads,51–53 in conjunction with long-read error correction54–56 and isoform characterization,57–59 will inevitably minimize the problem of chimeras in future studies. However, in the meantime, there are still many short-read datasets being generated, and there is a vast repertoire of archived short-read data that has been nearly two decades in the making.60 Thus, short-read RNA-Seq data yet possess scientific potential,61,62 and strategies improving its analysis are still relevant.\n\nIn relation to graph-based approaches, such as those implementing de Bruijn based strategies, including Trinity,63 rnaSPAdes64 and ABySS,65 as well as those creating networks based on splice sites identified following read mapping, such as TransComb,66 Cufflinks67 and StringTie,68 the ability to construct sets of contigs that represent the majority of transcripts present has been demonstrated.48–50,69,70 Potential has also been shown for graph-based approaches to provide detailed information on chimerism derived directly from the assembly process.40 During assembly, many graphs are constructed, and each aims to represent transcripts derived from single gene family.71 For graphs representing complex families, path choice during contig construction increases. This leads to the possibility of chimeras being produced in accordance with one of three broad categories: (i) over extension, (ii) increased sequence variation within regions and (iii) erroneously swapped regions.40,72 When reads are mapped to reference sets containing such chimeras, per-transcript counts values are affected.19,73 This increases the level of ambiguity within transcript expression profiles that are dependent on such counts.74,75 Additionally, it is these read count values that tools such as DESeq276 and EdgeR77 rely upon in order to characterize differential expression patterns. Given that short-read RNA-Seq platforms and de novo assembly software have been maturing for well over a decade,78,79 it is unfortunate that chimerism within assembled contigs, as well as the associated effects on read mapping and transcript expression profiling, have been under-characterized. A contributing factor is likely to be the difficulty in distinguishing between the extensive biological noise present within the transcriptome80,81 and artificial noise, including that of the erroneous chimerias created during assembly.72,82–84\n\nHere the effects that chimerism has on read mapping and differential expression analysis are explored using both simulated and real data. To aid this a tool called ChimSim, which takes a base set of reference transcripts and introduces a user-specified proportion of chimerism, was developed. Using ChimSim reads simulated off a pre-defined base set of reference transcripts, for example - one of the many species-specific cDNA libraries available from Ensembl,85 can be mapped to corresponding modified sets containing incrementing portions of chimerism, following which mapping success relative to each transcript present was measured. In this study transcripts present within the cDNA library of Drosophila melanogaster (fruit fly) were used as the base set. When reads are simulated to allow for multiple replicates divided into two conditions, one of which reflects a pattern of read over representation across one thousand randomly selected transcripts, the effects of chimerism on the identification of over-expressed transcripts can be explored. To do this, a differential expression experiment was performed iteratively, where within each iteration, the extent of chimerism present within the modified reference set used for mapping replicates within each condition, prior to analysis with DESeq2,76 was incremented. Finally, we generated an expectation of the relationship between modified base sets containing incrementing portions of chimerism and the underlying fruit fly base set from which the modified sets were created. At each increment, this relationship was summarized by r-squared values describing the correlation between alignment and base set transcript lengths relative to matches identified, using megablast,86 between the base set and modified set. The same metric was then obtained for contig sets assembled using three graph-based assemblers CStone,40 Trinity63 and rnaSPAdes,64 in relation to the underlying fruit fly cDNA base set from with the input reads were simulated. By comparison back to the background distribution created, involving explicitly defined levels of chimerism, an estimate of the extent of chimerism present within assembled contig sets across multiple replicates could be inferred. In relation to the latter, assemblies were also created, and r-squared values calculated for contigs produced from RNA-Seq reads sequenced from fruit fly, obtained from a study on alternative splicing.87 These too were compared to the background distribution describing the correlation between alignment lengths and base set transcript lengths at incrementing levels of chimerism.\n\n\nMethods\n\nChimSim is a tool that takes a set of sequences as input and creates an output set where a user-specified portion of the sequences is altered to be chimeric. ChimSim is written in Java and runs on operating systems with installed Java Runtime Environment 8.0 or higher (GNU General Public License v3.0). An executable version of ChimSim, as well as complete parameter descriptions, test data and source code, are available at: https://sourceforge.net/projects/chimsim/. An input reference set, for example, could consist of a set of expressed transcripts such as those available for varying species on Ensembl,85 or a compiled set of sequences manually defined by the user. Chimerism is introduced in accordance with one of the following three broad categories: (i) Over extension - transcripts have a region selected from a randomly chosen input transcript appended to them. The extent of over extension is selected at random from a range of between a minimum value (default: 100) and the length of the randomly selected transcript. (ii) Windowed Variation - windows are placed evenly along selected transcripts within which random variation is introduced. Window length (default: 200 nt), the number of windows (default: random between 1 and 5) and the level of divergence (default: 0.1, i,e, 10% of sites in window) can be defined by the user. If the length of a transcript is shorter than that of the combined length of the windows, then overlap is permitted. (iii) Window Shuffling - windows are created a similar manner to that described for (ii), but instead of variation being introduced, for each window a fragment of length equal to that of the window is selected at random from a different transcript and used to replace the region defined by a window. ChimSim outputs a text file containing the titles of transcripts selected to become chimeric, as well as the type of chimerism introduced.\n\nThe basic command to generate a set of modified transcripts from an input base set, where the default 10% of transcripts within the output will be chimeric and in accordance with default parameters, is: java -jar ChimSim.jar -ref_set path-to-ref-set.fasta.gz -gz true -out_dir path-to-out-dir. The command required to generate an output set of modified transcripts from transcripts within the input base set that range in length from 300 to 5000, and where 30% of the input transcripts within this range become chimeric (using window length of 250, a maximum of 10 windows and general divergence within windows of 0.4) would be: java -jar ChimSim.jar -ref_set path-to-ref-set.fasta.gz -out_dir path-to-out-dir -min_tln 300 -max_tln 5000 -chim 0.3 -tag -win_ln 250 -max_wins 10 -wgen_div 0.4. There are other ways in which a transcript can be made chimeric; for example, instead of a fixed window size, variable lengths could be used. Here the three categories that are most relevant to graph-based de novo assembly were included, but future developments may include increasing the number of categories available and allowing the user to select which they wish to apply.\n\nA base set of 26,680 transcripts containing all sequences ranging in length of between 300 and 5000 nt present within the fruit fly cDNA library (release-100 from Ensembl85) was created. This base set was used as a reference for simulating reads as required within subsequent sections as well as for the creation of modified base sets containing varying portions of chimerism. This base set along with the original cDNA library from which it was generated, as well as datasets used in subsequent sections describing differential expression analysis and contig creation have been provided as underlying data.88\n\nChimerism and read mapping\n\nTo explore the effects of chimerism on read mapping, the base set was used to create modified sets harbouring varying portions of chimerism. The portions of chimerism introduced ranged from 0% to 95% in steps of five. At each increment, ten replicates were performed. For each replicate five million read-pairs were simulated off the original base set and these were mapped to the modified set generated for that replicate. Mapping was performed with default parameters using Bowtie2,89 following which per-transcript read counts were obtained using the pileup.sh script of the bbmap package.90 R-squared value summarizing the correlation between mapped read count and transcript length were calculated using the R-package (version 4.1.1).91 Additionally, for each replicate of each increment, the total number of successfully mapped reads was recorded. Reads were of length 150 and simulated using CSReadGen (V0.1)92 in a similar manner to that described by Linheiro R & Archer J 2021,40 i.e., where insert size was 300 and no read error, background count variation (above normalized even coverage across all transcripts) or sequence divergence from the reference set was introduced. The approximate per site coverage provided by five million read-pairs across the base set was 26X. To compare the effect of chimerism on read mapping to that of random divergence between reads and the reference set being mapped to, the above was repeated but instead of introducing chimerism during iterations, ChimSim was used to introduce divergence using the -gen_div parameter. Random variation was increased from 0% to 25% in steps of one. For each of the ten replicates associated with a specified level of divergence the total number of reads mapped was counted.\n\nChimerism and differential expression\n\nTo explore the effects of chimerism on the detection of differentially expressed transcripts ten read datasets, each consisting of five million read-pairs, were simulated from the base set (See underlying data).88 These were allocated into conditions labelled as A and B. For the five datasets simulated for condition B, 1000 transcripts were selected at random, using the -rnd_ovr_exp parameter of CSReadGen, to have the required number of reads augmented by a factor of between one and five above the number needed to produce an even coverage across to all transcripts. For all transcripts the required number of read-pairs to produce an even coverage was allowed to vary by a factor of between 0.0 and 0.3, thus providing a level of background variation. Differential expression analysis was performed by obtaining per-transcript count values relative to the base set for each of the ten read datasets, as described in 2.1, and using these counts, in conjunction with their associated condition, as input for DESeq2.76 Within the latter the threshold for the identification of differentially expressed transcripts was a p-adjusted value of 0.05. Differential expression analysis was then repeated iteratively, where during each iteration ChimSim was used to create a modified base set within which a portion of the transcripts present were made chimeric. Reads were mapped to the modified set, counts obtained and DESeq2 employed as before. The portions of chimerism introduced ranged from 5% to 95% chimeric in steps of five. For each level of chimerism the number of differentially expressed transcripts identified between conditions A and B, were compared to these identified when using the unmodified base set for mapping. In addition, the overall presence of the 1000 transcripts specifically marked to have a higher possibility of being over-expressed was monitored as the level of chimerism increased.\n\nChimerism and de novo assembled contigs\n\nTo estimate the extent of chimerisim within de novo assembled contigs, the background relationship between the base set and modified sets, containing incrementing levels of chimerism, was characterized (See underlying data)88). The portions of chimerism introduced went from 5% to 95% in steps of five. Ten replicates were performed at each increment. For each replicate the megablast option86 of the BLAST+ package93 was used to identify the top match within the modified set to each transcript within the base set, and R-squared values summarizing the correlation between alignment length versus transcript length were calculated using the R-package (version 4.1.1). For each transcript within the base set the top ten hits were examined and the one with the longest continuous aligned region used. The distribution of these r-squared values across the incrementing levels of chimerism provided the background expectation for the metric of alignment length versus base set transcript length. Ten million read-pairs were then simulated off the base set, and CStone (v0.01),40 Trinity (v2.12.0)63 and rnaSPAdes (v3.11.1)64 were each used to assemble contigs. In a similar manner to before, megablast was used to compare base set transcripts, from which the reads were simulated, back to contigs and the R-package was used to calculate r-squared values. This procedure of simulating read-pairs from the base set, assembling them and calculating the r-squared values was repeated ten times. Values produced were compared to those calculated for the background relationship across incrementing levels of chimerism. In relation to real data, and as described by Linheiro R et al.,40 two adult fruit fly whole-body samples, from Pang et al. (2021) study on alternative splicing,87 were downloaded from NCBI SRA, study no. SRP297872; run number SRR13251053 for adult 1 and run no. SRR13251054 for adult 2. Reads were 100 nt in length and had been sequenced on Illumina’s Hi-Seq 2000 sequencer (See Underlying data88). Following quality filtering, using Trimmomatic (LEADING:10 TRAILING:10 SLIDINGWINDOW:4:15 MINLEN:36 ILLUMINACLIP:TruSeq3-PE.fa:2:30:10),34 they consisted of 31,543,384 and 29,812,987 read pairs. These were assembled using the three assemblers and megablast was used to compare transcripts from within the fruit cDNA library to the contigs produced. In this case the complete cDNA library was used and not the base set described for the simulations as sequenced reads could represent any transcript present, and not just those from which reads were simulated. As before, the R-package was used to calculate r-squared values summarizing the correlation between alignment length versus transcript length.\n\n\nResults and discussion\n\nFor a single replicate, Figure 1 depicts the relationship between transcript lengths and read counts once reads were mapped to the base set, containing 0% introduced chimerism, from which they were simulated. The inset table contains the r-squared value calculated for the line of best fit, as well as those obtained for the other nine replicates. The high values confirm that reads were simulated as expected when specifying even transcript coverage, no background variation, and no read error. When reads simulated in this manner are mapped to modified base sets containing incrementing levels of chimerism (Figure 2A), a progressive lowering of the r-squared values occurs. However, at 95% chimerism a strong correlation remains, the lowest value from ten replicates being 0.8141. R-squared values calculated for just the chimeric transcripts within each set (Figure 2B) are within the range of 0.8158 to 0.8638 across all increments. This is consistently lower than the values calculated for the non-chimeric transcripts (Figure 2C) which indicates that when all transcripts are present, it is the presence of the chimeric ones that lowers the overall values. When r-squared values obtained using transcripts associated with each individual category of chimerism are plotted (Figures 3A to C), the over-extension category had less of an effect than those of windowed variation and window shuffling. In relation to the overall number of reads mapped (Figure 4A), although mapping sucess decreases as chimerism is increased, the lowest point occurs for a replicate at 95% chimerism, when 77% of reads were still mapped. The combination of these results indicated that, even when faced with extreme chimerism, on a surface level read mapping does not appear to perform poorly when looking at basic count values. This is because much of the variation introduced by chimerism is not novel and the majority of reads will find a transcript to map to. Importantly, this suggests that there could be a hidden impact of chimerism on downstream data analysis that is hard to predict based on mapped read counts alone. Figure 4B indicates that this is not the case for the introduction of random variation between the reference set and the reads being mapped, where a rapid decline in mapping success is evident.\n\nReads containing no sequencing error, and distributed evenly across all transcripts, were mapped to the base set (containing 0% introduced chimerism) from which they were simulated. The x-axis indicates the transcript length, and the y-axis indicates the number of mapped reads. The r-squared value associated with the line of best fit is indicated within the inset table (in red). The other r-squared values within the table indicate those associated with the other nine repetitions of read simulation and subsequent mapping to the base set.\n\n(A) R-squared values (y-axis) summarizing the correlation between transcript length and mapped read count in relation to reads simulated off the base set but that are mapped to related sets containing incrementing levels of chimerism (x-axis). At each increment of ten replicates were performed. (B) Same as for (A) but only the chimeric transcripts, at the indicated increment of chimerism, within the modified set are included. (C) Same as for (A) but only the non-chimeric transcripts, at the indicated increment of chimerism, within the modified set are included. In all cases, the medians are shown within each box. Whiskers extend to the furthest data point that is within 1.5 times the interquartile range and points beyond this are outliers (black circles).\n\nR-squared values (y-axis) between the length of individual transcripts (to which chimerism had been introduced) and mapped read count, i.e., those presented in Figure 2B, were divided into the three categories of chimerim implemented within ChimSim. These were: (A) Over-extension, (B) Window variation and (C) Window shuffling. In all cases the medians are shown within each box. Whiskers extend to the furthest data point that is within 1.5 times the inter quartilerange and points beyond this are outliers (black circles).\n\nReads simulated off the base set were mapped to: (A) modified base sets containing incrementing levels of chimerism (x-axis) following which the total number of reads successfully mapped counted (y-axis) and (B) modified base sets containing incrementing levels of sequence divergence (x-axis) and the total number of reads successfully mapped counted (y-axis). In panel (A) the red line indicates the lowest mapped read percentage achieved across all replicates and increments. In all cases the medians are shown within each box. Whiskers extend to the furthest data point that is within 1.5 times the interquartile range and points beyond this are outliers (black circles).\n\nIn Table 1 it is observed that when datasets belonging to conditions A and B were mapped to the base set containing 0% introduced chimersim, and differential expression analysis performed, 2853 and 400 transcripts were identified as being over- and under-expressed. Of the 2853 over-expressed transcripts, 980 were within the set of 1000 transcripts randomly selected for increased representation within condition B. The remaining 1873 being a consequence of the random background variation applied. The other rows of Table 1 show that despite chimera’s having a relatively low effect on general mapping success, i.e., Figures 2 and 4, increasing the level of chimerism within the base set prior to mapping and subsequent differential expression analysis has a large effect on the identification of differentially expressed transcripts. Of the 980 transcripts that were identified as being over-expressed, that belonged to the set of 1000 transcripts selected increased read representation within condition B, the number detected at each incrementing level of chimerism rapidly diminished. Likewise, but more generally, Figure 5 indicates that for each 5% increment in chimerism, the number of overall transcripts detected as being over- and under-expressed that agree with those identified in the absence of chimerism (Table 1 - row 1) also diminishes. This highlights the ambiguity that can be introduced during downstream data analysis as a result read mapping yielding unreliable, not necessarily diminished, counts when faced with chimerism.\n\nThe red text indicated row one, the numbers obtained when using a reference set containing no introduced chimerism. The last column indicates the number of transcripts identified as being over-expressed that were within the set containing the 1000 transcripts randomly selected for increased read representation within condition B during read simulations, i.e., increased chance of over-expression.\n\nTen paired-read datasets were simulated and divided evenly into two conditions. Unlike previous simulations, per-transcript read representation was allowed to vary. Additionally, within one of the conditions, 1000 transcripts were over-represented across the five replicates. Differential expression analysis between the two conditions was performed, using the non-chimeric base set, in order to obtain a list of over- and under-expressed transcripts. Differentially expression was then iteratively repeated in a similar manner, but where the extent of chimerism within the reference set used was incremented (x-axis). The middle grey section of each bar represents the number of (A) over- and (B) under-expressed transcripts identified at the indicated level that were also identified when performing differential expression analysis using the non-chimeric base set. In both panels the dark grey area of each bar indicates transcripts that were identified as being differentially expressed solely when using the non-chimeric reference set. The corresponding light grey bar represents transcripts identified as being differentially expressed when solely using a modified reference into which the indicated level of chimerism was introduced. The red lines in both panels indicate the total number of over- and under- expressed transcripts identified when using the non-chimeric base set.\n\nFor the three assemblers, Figure 6A displays the r-squared values that describe the correlation between alignment lengths and contig lengths. The alignments used were those between base set transcripts and best matching contigs. Higher values indicate that larger portions of the contigs were aligned, thus indicating better assemblies. The values obtained, ranging from 0.8832 to 0.9591, suggest that as a whole contigs produced by all assemblers reflected well the regions of the base set transcripts to which they aligned. Figure 6B shows the distribution of r-squared values describing the equivalent correlation but, instead of assembled contigs, modified base sets of transcripts containing varying levels of chimerism were used. Direct comparison to the equivalent values obtained for the three assemblers (Figure 6A) suggests that the level of chimerism within the assembled contigs could be in the range of between 5-15%. Figure 6C shows that the r-squared values obtained for contigs assembled from RNA-Seq data obtained from the two fruit fly whole adult samples are only slightly lower than those from the simulated datasets and thus the expected level of chimerism would not be dissimilar. Given the effects that such sequences have on differential expression analysis, even at the 5-15% levels (Table 1 and Figure 5), the analysis performed for Figure 6 highlights the need to either quantify these sequences further during the de novo assembly process or to circumvent the problem of chimerism completely by moving towards approaches that utilize long read technologies.\n\n(A) For the contigs created by each assembler, across each of ten replicates, r-squared values describing the correlation between alignment length and contig length were calculated. The alignments used were those between the top contig match for each transcript within the base set from which reads were simulated. The red dotted lines indicate the maximum and minimum value obtained. (B) The distribution of r-squared values describing the equivalent correlation described in (A), but instead of assembled contigs, modified base sets of transcripts containing varying levels of chimerism were used. The dotted red line indicates where the maximum and minimum values obtained for the three assemblers indicated within panel A could be projected on-to the x-axis. (C) Following quality filtering of two adult fruit fly whole-body samples from Pang et al. (2021),40 consisting of 31,543,384 and 29,812,987 read pairs, reads were assembled using each of the three assemblers and the r-squared values described for (A) calculated. In all cases the medians are shown within each box. Whiskers extend to the furthest data point that is within 1.5 times the interquartile range, and points beyond this are outliers (black circles).\n\nFigure 7A indicates the range of transcript lengths present within the modified base set created across each replicate used in Figure 6B. The slight increase observed with incrementing chimerism is as a result of the transcripts selected for over-extension. Despite this minimal increase in the overall length distributions, Figure 7B indicates that the number of base set transcripts being represented by a match within the modified sets is rapidly reduced with incrementing chimerism. When 5% of the transcripts within the modified sets are chimeric the median number of transcripts within the base set finding a megablast match is 22495, whilst at the 15% level of chimerism it is 194343. This is due to sequence variation associated with chimerism increasing. Although preliminary, this suggests that the extent of chimerism within de novo assembled contigs will also have an effect on annotation tools that rely on searches based on sequence similarity.\n\n(A) Lengths (y-axis) of transcripts within the modified base sets following the introduction of chimerism to varying degrees (x-axis). The numbers along the top of each box and whisker indicate the number of transcripts above 5000 nt in length (red line). (B) The number of base set transcripts (y-axis) finding a representative match within the modified base sets containing incrementing degrees of chimerism. Despite the consistency in modified base set transcripts lengths as chimerism is introduced (panel A), the number of base set transcripts represented rapidly diminishes. In all cases the medians are shown within each box. Whiskers extend to the furthest data point that is within 1.5 times the interquartile range and points beyond this are outliers (black circles).\n\n\nConclusion\n\nAlthough it is known that the de novo assembly of short-read RNA-Seq data can result in chimeric contigs, the extent of such chimerism has been poorly quantified, as has the effects that such chimerism has on data analysis. In this study we have demonstrated these effects on read mapping and on the identification of differentially expressed transcripts. We have also indicated to what extent such chimerism could be expected within contigs assembled using three graph-based assembly tools. Despite all tools performing well, the rapid consequence of even low levels of chimerim on results interpretation, indicate that further effort is required to include information relevant to chimera quantification, and that results dependent on short-read assembly must be present within the context of this information. An inability to make this improvement to current assemblers would suggest that transcriptomics experiments must strive to move away from using short-read data. If not the consequences on scientific robustness in relation to results-base conclusion will be difficult to mask.\n\n\nData availability\n\nZenodo: Quantification of the effects of chimerism: datasets\n\nDOI: 10.5281/zenodo.587792288\n\nThis project contains the following underlying data:\n\nBaseSetTranscripts.zip: Contains a file with all transcripts present within the Ensembl release-100 of the fruit fly cDNA library and another file containing sequences ranging in length of between 300 and 5000 nt from that cDNA library\n\nDEReads.zip: Contains the ten sets of paired reads, divided into conditions A and B as indicated, that were used for differential expression analysis.\n\nDEChimSimRefs.zip: contains the references sets harbouring varying levels of chimerism that were used for differential expression analysis.\n\nDeNovoAssemblies_SimulatedData.zip: Contains the de novo assemblies.\n\nReads_RealData_WholeBody_1.zip: Contains the whole body read datasets from adult fruit fly 1 following quality filtering.\n\nReads_RealData_WholeBody_2.zip: Contains the whole body read datasets from adult fruit fly 2 following quality filtering.\n\nDeNovoAssemblies_RealData.zip: Contains the assemblies generated when using the previous two datasets as input.\n\nAll data is available under the terms of the Creative Commons Attribution 4.0 International license\n\n\nAuthor contributions\n\nJohn Archer: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft. 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PubMed Abstract \| Publisher Full Text\n\nBushnell B: BBMap: A Fast, Accurate, Splice-Aware Aligner. Conference: 9th Annual Genomics of Energy & Environment Meeting.2014. Publisher Full Text\n\nTeam RC. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2020. Reference Source\n\nArcher J: CSReadGen website.2020. Reference Source\n\nCamacho C, Coulouris G, Avagyan V, et al.: BLAST+: Architecture and applications. BMC Bioinform. 2009; 10. PubMed Abstract \| Publisher Full Text" }	[ { "id": "121628", "date": "15 Feb 2022", "name": "Ben J. Mans", "expertise": [ "Reviewer Expertise De novo transcriptomics", "proteomics used for validation of de novo transcriptomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors investigated the artefact of chimerism that may occur during de novo assembly using short-read assemblers. This is an important area within the field of de novo transcriptome assembly since it may result in erroneous transcripts as well as affect estimates of differential expression and transcript abundance. Chimerism was investigated by simulating the formation of various levels of chimers (5-95%) and evaluating how this affects mapping results and correlation to a reference set. It is shown that significant levels of reads can be mapped to datasets with high percentages of chimeric sequences and that this is true for various forms of chimeric sequences including over-extension (end fusion), variation within a sequence, and fragment exchange within transcripts. It is also shown how the level of chimeric sequences can impact the detection of over/under-expressed transcripts with less over-expressed transcripts detected at higher chimerism. De novo assemblies were also performed using raw original data and it was estimated that de novo assemblies may contain as much as 5-15% chimeric sequences. The study concludes that de novo transcriptome sequencing should move away from using short-read data for transcriptome assembly. The study is well described and the results and conclusion seem warranted. It highlights an important aspect in transcriptome sequences that researchers in the field should be aware of. Issues follow below.\nGeneral: The observation that chimeric sequences form during transcriptome assembly is not new and several programs deal with this and actively remove chimeric sequences. Awareness of over-extension also allows identification of over-extended fragments that can be trimmed from transcripts (for example if proteins have secretory peptides and these can be correctly identified). Research on how to detect and remove chimeric sequences without losing bona fide transcripts should therefore also be an important consideration of future studies. In the current study, I did not detect much regarding existing means to detect and remove chimers and this may be something the authors can address.\nGeneral: One of the aims of transcriptome sequencing would be to obtain coding sequences for genes as opposed to transcripts alone. Extraction of open reading frames may result in a number of outcomes, such as loss of over-extension, or truncation of open reading frames due to window shuffling or variation since the formation of chimeric sequences does not guarantee the conservation of an intact open reading frame. Even with the estimated 5-15% chimeric sequences that may be present in de novo transcriptomes, the use of open reading frames could negate some of the problems of chimerism, or help to identify and remove chimeric sequences. Could the authors comment on how many of the simulated chimeric sequences would result in intact open reading frames, or if analyzed using downstream analysis methods such as conserved domain prediction, how many would be discarded from analysis since they do not yield intact domains. It may be that the majority of chimeric sequences are removed during quality curation and would then not pose a major impediment in transcriptome assemblies.\nFigure 1: This figure nicely shows the correlation between mapping coverage and transcript length for the reference set with very high correlation between mapped reads and transcript length. It is not clear from the methods whether the read count has been normalized (TPM, RPKM), so to find such good correlation is quite interesting, since in which real transcriptome data you may find that small transcripts may be highly abundant, or that large transcripts may have low abundance. I assume that this is because the simulated paired reads are normalized. Would this mean that the correlation method used here can not be used on real data to estimate chimeric levels? Would this explain to some extent the variation in correlation in Figure 6 that may not necessarily be due to chimeric sequences?\nFigure 6: Based on the correlation it seems as if Cstone (an assembler by the authors) do much better than other assemblers. While this is somewhat implied, the authors do not explicitly state that their assembler performs better than the others. Does Cstone result in less chimeric sequences? If so, it would also be of interest to provide alternative measures of quality such as the number of intact open reading frames. I suspect that if open reading frames are extracted and the same mapping correlation determined, that the curves for the chimeric sequences may be deeper since less open reading frames may be obtained compared to the reference set.\nPage 3: Drosophila in italics.\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": "7999", "date": "24 Mar 2022", "name": "John Archer", "role": "Author Response", "response": "\"The authors investigated the artefact of chimerism that may occur during de novo assembly using short-read assemblers. This is an important area within the field of de novo transcriptome assembly since it may result in erroneous transcripts as well as affect estimates of differential expression and transcript abundance. Chimerism was investigated by simulating the formation of various levels of chimers (5-95%) and evaluating how this affects mapping results and correlation to a reference set. It is shown that significant levels of reads can be mapped to datasets with high percentages of chimeric sequences and that this is true for various forms of chimeric sequences including over-extension (end fusion), variation within a sequence, and fragment exchange within transcripts. It is also shown how the level of chimeric sequences can impact the detection of over/under-expressed transcripts with less over-expressed transcripts detected at higher chimerism. De novo assemblies were also performed using raw original data and it was estimated that de novo assemblies may contain as much as 5-15% chimeric sequences. The study concludes that de novo transcriptome sequencing should move away from using short-read data for transcriptome assembly. The study is well described and the results and conclusion seem warranted. It highlights an important aspect in transcriptome sequences that researchers in the field should be aware of. Issues follow below.\" We thank the reviewer for this great review of our manuscript. The points raised were valuable additions and will be discussed further within the next version. Once all other reviewer comments are in we plan on working on this. In the meantime we are briefly commenting here on these specific responses as they were provided some time ago and progress on version preparation has been slow. \"General: The observation that chimeric sequences form during transcriptome assembly is not new and several programs deal with this and actively remove chimeric sequences. Awareness of over-extension also allows identification of over-extended fragments that can be trimmed from transcripts (for example if proteins have secretory peptides and these can be correctly identified). Research on how to detect and remove chimeric sequences without losing bona fide transcripts should therefore also be an important consideration of future studies. In the current study, I did not detect much regarding existing means to detect and remove chimers and this may be something the authors can address.\" The context of our study was to highlight the dangers of chimeras introduced during the de novo assembly of short-read data, specifically using a graph-based assembly approach. Previously we have discussed in detail how such chimeras are introduced, as well as created an assembler that identifies chimeric contigs based on the structural complexity of the underlying graphs from which they were derived; reference [40] in manuscript (Linheiro R, Archer J: CStone: A de novo transcriptome assembler for short-read data that identifies non-chimeric contigs based on underlying graph structure. PLoS Comput. Biol. 2021; 17: e1009631). The reviewer is correct in indicating that there are existing tools that attempt to identify chimeras within sets of contigs post assembly, but our analysis is sitting in the realm between immediate post-assembly and prior to downstream processing (and associated usage of such tools). We are specifically interested in the problem of short-read assembly itself and we are largely attempting to highlight this. In relation to the more generic removal of chimeras from sets of sequences using third party tools, there are various methods that can be applied, ranging from extracting open reading frames (as subsequently suggested by reviewer) to the comparison of contigs to varying databases of known non-chimeric transcripts, but no consensus exists on the optimal method and all have limitations in what they can achieve. Related and more importantly here, the nature of chimeric contig creation during de novo assembly, combined with implicit complexity of “genuine” isoform variation, creates potential for a continuous spectrum of chimerism ranging from non-chimeric sequences to complete chimeric “junk”, and passing through various levels of partial chimerism of varying type along the way. Heuristic approaches for third party chimeric contig removal, although inevitability improving downstream analysis results to some extent, cannot guarantee compete non-chimerism nor can it highlight the variation associated with such chimerism on end results. Bearing in mind that at times hundreds of thousands of contigs are produced by de novo assemblers that are aimed at representing the fewer tens of thousands of genuine expressed transcripts, in some cases it can be impossible to know whether or not a particular contig is chimeric or a true representation of a previously uncharacterized isoform. Statistical approaches can correct to a certain extent, but they cannot completely undo the implicit flaws associated with heuristic assembly algorithms when faced with such a complex problem of accurately assembling RNA-Seq short-read data using graph structures. This issue must be acknowledged. In our study, we were aiming to show such effects across a wide uncorrected spectrum of chimerism, just after the assembly process (from reads simulated from a base set reference), where the base set was then subsequently used with incrementing levels of chimerism during mapping and differential expression analysis. Given this scope, we feel that a complete review/benchmark of correctional approaches was beyond what we were hoping to highlight, but we agree that more on this point should be mentioned within the manuscript. As such when we produce a revised version we will incorporate more on this relevant topic. \"General: One of the aims of transcriptome sequencing would be to obtain coding sequences for genes as opposed to transcripts alone. Extraction of open reading frames may result in a number of outcomes, such as loss of over-extension, or truncation of open reading frames due to window shuffling or variation since the formation of chimeric sequences does not guarantee the conservation of an intact open reading frame. Even with the estimated 5-15% chimeric sequences that may be present in de novo transcriptomes, the use of open reading frames could negate some of the problems of chimerism, or help to identify and remove chimeric sequences. Could the authors comment on how many of the simulated chimeric sequences would result in intact open reading frames, or if analyzed using downstream analysis methods such as conserved domain prediction, how many would be discarded from analysis since they do not yield intact domains. It may be that the majority of chimeric sequences are removed during quality curation and would then not pose a major impediment in transcriptome assemblies.\" As the reviewer points out the removal of chimeric contigs based on open reading frame identification is an effective way to remove many chimeric contigs. However, we feel that this point must be taken into context with the issue mentioned in the previous paragraph as: (i) due to the continuous spectrum that describes the overall extent of chimerism within a de novo assembled dataset (including that of redundant portions of transcripts), there can be still many sequences that pass this filter but are still chimeric in some way (and so affect read counts used in downstream analysis figure 2) and (ii) when contigs at the end of the spectrum that are closer representations of true transcripts are compared to databases containing what we believe are correct sequences, extremely close matches can be found making it impossible to distinguish subtle chimeric forms. Within the latter patterns between co-evolving sites or these between recombinant breakpoints can be obscured, as can read counts but likely to a lesser degree. Nonetheless the latter will still have an effect on the end result of downstream analysis. Effectively graph-based de novo assembly of short-read RNA-Seq data produces a wide range of sequence representations of the underlying transcripts of varying quality, and generally massively over represents the number of true transcripts expressed. Methods of filtering to reduce the extent of chimerism clearly improve these initial datasets, but chimeras will still be present in unknown quantities. Once again here we are attempting to demonstrate the effects of chimerism on downstream analysis across a wide range of chimerism, prior to filtering of the contigs. Our main point is that their presence has an effect on analysis and quantification of this presence is unreliable, therefore we just show the entire spectrum. It is clear from the reviewer comment that there is a requirement to emphasise this more within the manuscript and we will aim to do so. \"Figure 1: This figure nicely shows the correlation between mapping coverage and transcript length for the reference set with very high correlation between mapped reads and transcript length. It is not clear from the methods whether the read count has been normalized (TPM, RPKM), so to find such good correlation is quite interesting, since in which real transcriptome data you may find that small transcripts may be highly abundant, or that large transcripts may have low abundance. I assume that this is because the simulated paired reads are normalized. Would this mean that the correlation method used here can not be used on real data to estimate chimeric levels? Would this explain to some extent the variation in correlation in Figure 6 that may not necessarily be due to chimeric sequences?\" Figure 1 was aimed at verifying the read simulation process, where reads were simulated from a base reference set of transcripts, and the main parameters specified were: even read coverage across transcripts within the input reference set (i.e. required read counts normalized by transcript length), 0% per site error, no background variation and 0% chimerism. The plots of mapped read count versus transcript length, and the associated r-squared values, were to explicitly confirm that reads were simulated in this manner. There is no sequencing process (or assembly) involved here, just reads directly simulated off the underlying base reference set (where read numbers required to represent each transcript were directly proportional to the length) and then those reads mapped back against the reference transcripts from which they were simulated from. Given this scenario, in figure 1 if the raw count does not correlate directly with reference transcript lengths then the simulation process did not go as expected. However the R-squared values in the figure show that the simulation process did perform as expected relative to the described parameters. Reads simulated in this manner where subsequently used in experiments where increasing levels of chimerism were introduced into the base reference set (post simulation, but pre-mapping) in order to study the effect of chimerism on the counts obtained. In figure 2 reads simulated in this manner are mapped to modified reference sets containing incrementing levels of chimerism, and the r-squared values between contig length and read counts plotted. This is to demonstrate that incrementing levels of chimerism within the reference set used during mapping (of reads derived from the non-chimeric set) have an effect on the counts obtained following each increment of chimerism. Given a read dataset, and some hypothetical associated representative reference set of transcripts, the reviewer is correct in observing that a correlation between read count and contig length could not be used accurately used to estimate levels of chimerism – but in figure 2 this was never our intension. However, in figure 6 which is referred to by the reviewer, the comparison is different. In figure 6 we are not comparing read counts. We are comparing the correlation between contig lengths and base set transcript lengths where the contigs were: (a) assembled from simulated reads, (b) base set transcripts themselves but with incrementing levels of chimerism introduced and (c) assembled from real RNA-Seq short-read data derived from fruit fly. In this case we are not saying that the correlation is an exact indication of chimeric content, but that when using (b) as a background expectation, it is a reasonable “hint” on the expected level. In the next version of the manuscript we will clarify this further. \"Figure 6: Based on the correlation it seems as if Cstone (an assembler by the authors) do much better than other assemblers. While this is somewhat implied, the authors do not explicitly state that their assembler performs better than the others. Does Cstone result in less chimeric sequences? If so, it would also be of interest to provide alternative measures of quality such as the number of intact open reading frames. I suspect that if open reading frames are extracted and the same mapping correlation determined, that the curves for the chimeric sequences may be deeper since less open reading frames may be obtained compared to the reference set.\" The paper where we describe and benchmark CStone is effectively a predecessor of this study. In that paper, and here, importantly, we never claim that CStone performs better than the other two assemblers used. This is because firstly, the aim of CStone’s development was to simply implement an approach to contig construction using a graph-based method of assembly, where the structure of underlying graphs could be used to flag individual contigs as being non-chimeric (if sufficiently few paths exist). It was never intended to be an improvement on similar graph-based short-read de novo assembly approaches, just to produce comparable contigs so that a demonstration of obtaining this extra information on chimerism could be achieved. In other words, CStone is a tool that does not use an array of contig filtering packages to optimize the end result following graph-based contig construction, but it does clearly demonstrate that information derived from graph structures can have relevance to the interpretation of the results from downstream analysis, and that the contigs produced are comparable to other state-of-the-art tools to make this demonstration convincing. Our aim was to widely encourage assembly tool developers to incorporate such output in an accessible manner. Secondly, the correlation between contig length and transcript length displayed in figure 6 is not a sufficient metric to claim an improvement on assembly. Yes, the stronger this correlation is, the more closely related in length the contigs and representative reference set transcripts are, but there are many other factors involved, for example, the quality of the underlying reference set used for comparison, the divergence of this reference set from the input reads, the success of identifying open reading frames (as mentioned by reviewer) and the number of true transcripts actually represented. To approach making such a claim even of an improved assembly all base parameters, such as k-mer size, would need to be analysed in relation to an array of different organisms. With this in mind we have previously shown that CStone is approximately 10% less sensitive at detecting some transcripts (reference [40] in manuscript). The reviewer is absolutely correct here in suggesting that if open reading frames are extracted and the same mapping correlation determined, then the curves for the chimeric sequences may be deeper (or at least different) since fewer open reading frames may be obtained compared to the reference set. Our interest was more in relation to the assembly process, and highlighting the variation in end results that can be directly dependent on this, and we have not performed this specific analysis of testing reading frames. It is something that we could be open to doing so in the future, but it should be noted that in both this paper and in our CStone paper we are suggesting that long read sequencing technologies are the future, and we need to be careful on how we direct our time. This type of analysis would have been very nice to see in relation to short-read assembly, widely highlighted, perhaps ten years previously. Within the next version of the manuscript we will make a point to highlight this concern in relation to the open reading frames and figure 6." } ] }, { "id": "137647", "date": "07 Jun 2022", "name": "Kun Lu Lu", "expertise": [ "Reviewer Expertise Plant 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 authors use two types of transcript sets as reference, including one base set and several modified sets from the fruit fly cDNA library, to explore the effects of varying portions of chimerism on reads mapping, differential expression analysis, and annotation under simulating scenario. Also, the authors estimate chimerism extent within assembled contigs created by three assemblers. It’s vital work for de novo assembly of short-read RNA-Seq data.\nMajor: 1. The authors only demonstrated the effects of chimerism for one species, fruit fly; However, whether the same results existed in other animals, e.g. mouse or plants, e.g. Arabidopsis thaliana.\n2. The chimerism extent within assembled contigs created by three assemblers is in the range of 5~15%, of which CStone shows better assemblies. The authors should explain the reason which leads to the difference between these assemblers. In addition, the authors should quantify the effect from three broad categories of chimeras within assembled contigs created by three assemblers instead of overall chimerism extent.\n3. Erroneous chimeric contigs created by assemblers maybe result in poor results, however, chimeric RNA sometimes referred to as a fusion transcript, which can be expressed somewhere, hence, how to distinguish erroneous chimeric contigs from all chimeric contigs?\n\nMinor: Introduction section: 4. “Drosophila melanogaster” should be italic\nMethod section: Chimerism and read mapping 5. The statement part is cumbersome, e.g. “for each replicate of each increment, the total number of successfully mapped reads was recorded” and “For each of the ten replicates associated with a specified level of divergence the total number of reads mapped was counted”.\nMethod section: Chimerism and differential expression 6. The adjusted method of P value and fold change of expression level should be listed\nResults and discussion 7. Figure 1, the unit of plot axis are unknown, like million for mapped reads if using raw counts, bp for transcript length.\n8. Table 1, how to define over-expressed transcripts and under-expressed transcripts?\n9. Figure 5, please add legends for different colors in this figure.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "8501", "date": "12 Jul 2022", "name": "John Archer", "role": "Author Response", "response": "“Reviewer summary: The authors use two types of transcript sets as reference, including one base set and several modified sets from the fruit fly cDNA library, to explore the effects of varying portions of chimerism on reads mapping, differential expression analysis, and annotation under simulating scenario. Also, the authors estimate chimerism extent within assembled contigs created by three assemblers. It’s vital work for de novo assembly of short-read RNA-Seq data.” We thank the reviewer for taking the time to review our manuscript and for recognizing the often overlooked importance of quantifying the effects of chimerism on downstream RNA-Seq data analysis. Below we will respond briefly to each of the major comments. These comments, as well as those from reviewer 1, will be incorporated into the next version of our manuscript that we will begin working on. The comments have added greatly to the clarity our work. -------- “Reviewer major comment 1: The authors only demonstrated the effects of chimerism for one species, fruit fly; However, whether the same results existed in other animals, e.g. mouse or plants, e.g. Arabidopsis thaliana.” We used the fruit fly transcript reference set as a starting point in order to simulate subsequent modified transcript reference sets containing varying degrees of chimerism, where the chimeras introduced fell into three distinct computationally generated previously described categories: (i) over extension, (ii) increased sequence variation within regions and (iii) erroneously swapped regions. What we have not done is explore the extent of each of these individual categories, or other types of chimerism, across multiple different species. Our aim was solely to demonstrate the effects of chimera presence on downstream RNA-Seq data analysis regardless of species. Given that we were introducing predefined types and proportions of chimerism the starting reference library could have been from any species. We choose fruit fly as it is a model organism and we felt that the quality of the starting transcripts would be generally higher. An alternative could have been to start with a fully simulated library of transcripts that did not represent any individual species. However this is a very interesting point raised by the reviewer related to the nature of chimerism within assemblies. For our analysis, in order to explore the effects of chimerism in general on downstream analysis, we use a set probability for the proportion of each category of chimerism to be created within the general set of transcripts selected to be made chimeric; the latter being based on a percent of the total number of transcripts. However and related to the reviewer’s point, data derived from different species could have the possibility of being more prone to particular types of chimerism, beyond the three simple types we have applied. The main problem is that the extent of each category of chimerism within assembled data from different species would be very difficult, if not impossible, to quantify given that definitive sets of correct non-chimeric transcripts do not exist as a prior. This is why we choose to introduce defined levels of simulated chimerism that are species independent; despite using the fruit fly cDNA library as a seed. For example, in relation to de novo assembled contigs representing RNA-Seq data from a hypothetical species A, where the general diversity can be summarized by fewer kmers of given length relative to a hypothetical species B, prior to graph construction there may be increased numbers of kmers repeated between isoforms derived from a single gene family, or from different gene families. If an increase in shared kmers does occur, then more chimeras would be expected during assembly. This is because the de Bruijn graphs used during short-read assembly are a representation of the connectivity between these kmers and if more are shared between families, or regions families, increased numbers of chimeric paths across graphs would exist. Thus, the quantity of chimeras present would be influenced by transcriptome diversity that is species dependent. However, in simulated data this is not the case as all parameters are clearly defined during chimera introduction and we can simply observe the effects of their presence at varying percentages, relative to the starting base set. Our point on the general effects of pre-defined chimerism is highly relevant to RNA-Seq data analysis and we hope that our initial reply to this comment will convince the reviewer that the very interesting point raised is one that is exceptionally difficult to approach and is beyond the scope of what we were attempting to achieve with our manuscript. We will highlight this further within the next version of the manuscript. -------- “Reviewer major comment 2: The chimerism extent within assembled contigs created by three assemblers is in the range of 5~15%, of which CStone shows better assemblies. The authors should explain the reason which leads to the difference between these assemblers. In addition, the authors should quantify the effect from three broad categories of chimeras within assembled contigs created by three assemblers instead of overall chimerism extent.” CStone did appear to have less chimeras than the other two assemblers used, and this is likely due to a reduced tendency to construct overly long contigs. During benchmarking when CStone was run on short-read data generated using transcripts with a maximum length of 5000 bp from fruit fly, leopard, rat and canary cDNA libraries [1], the numbers of contigs above 5000 bp assembled were 6, 2, 3 and 2. When the other two assemblers were used to assemble the same data the numbers of contigs above 5000 bp for these species were 126, 21, 72 and 19 (rnaSPAdes) and 464, 113, 219 and 211 (Trinity). When run on real data similar patterns were observed. It is very likely that the longer, and at times overextended, contigs contain more chimeras. However, CStone was also shown to be slightly less sensitive at detecting transcripts and was intended as a tool to demonstrate that it is possible to output chimera information based on the underlying graphs structures used during the assembly process. For this reason, we would not say that CStone performed better – just that it conservatively defined a minimum range of the extent of chimerism. We will clarify this further with the next version of the manuscript. -------- “Reviewer major comment 3: Erroneous chimeric contigs created by assemblers maybe result in poor results, however, chimeric RNA sometimes referred to as a fusion transcript, which can be expressed somewhere, hence, how to distinguish erroneous chimeric contigs from all chimeric contigs?” This is another reason why we were using simulated “known” chimeras, that we could flag as being artificially chimeric as they were created by the ChimSim tool. At times, it is not possible to separate correctly assembled transcript representing “fusion transcript”(s) from those erroneously introduced during assembly. Our aim is to highlight the general effects of the latter, but not necessarily be able to identify them within assemblies. If they could be identified and removed with certainty of being erroneously introduced, RNA-Seq data analysis would be in a very good place; and it would also largely remove the need to quantify their effects. We will add this discussion to the next version of our manuscript. -------- “Reviewer minor comments:” various Each of the minor comment provided will be incorporated into the next version. References 1. Linheiro R, Archer J. CStone: A de novo transcriptome assembler for short-read data that identifies non-chimeric contigs based on underlying graph structure. PLOS Comput Biol. 2021;17: e1009631. doi:10.1371/JOURNAL.PCBI.1009631" } ] } ]	1	https://f1000research.com/articles/11-120
https://f1000research.com/articles/10-1008/v1	05 Oct 21	{ "type": "Systematic Review", "title": "The implications of Industry 4.0 on supply chains amid the COVID-19 pandemic: a systematic review", "authors": [ "Mohammad Nurul Hassan Reza", "Sreenivasan Jayashree", "Chinnasamy Agamudai Nambi Malarvizhi", "Md Abdur Rauf", "Kalaivani Jayaraman", "Syed Hussain Shareef", "Mohammad Nurul Hassan Reza", "Chinnasamy Agamudai Nambi Malarvizhi", "Md Abdur Rauf", "Kalaivani Jayaraman", "Syed Hussain Shareef" ], "abstract": "Background: COVID-19 has caused significant disruptions in supply chains. It has increased the demand for products and decreased the supply of raw materials. This has interrupted many production processes. The emerging technologies of Industry 4.0 have the potential to streamline supply chains by improving time-sensitive customized solutions during this emergency. Purpose: This research examines the effects of the epidemic on supply chains and how these effects are reduced through Industry 4.0 technology. Design/methodology/approach: An extensive literature review using the “Preferred Reporting Items for Systematic Review and Meta-Analysis” method was carried out on the impact of the COVID-19 pandemic on supply chains and Industry 4.0 technologies. The study was undertaken by selecting keywords validated by experts and a search was conducted in the Scopus, ProQuest, and Google Scholar databases. Publications from the leading journals on these topics were selected. The bibliographical search resulted in 1484 articles followed by multiple layers of filtering. Finally, the most pertinent articles were selected for reviewing, and a total of 53 articles were analysed. Findings: This study discusses the impact of COVID-19 on the supply chain and how the emerging technologies of Industry 4.0 can help manufacturers to ease the impact. These technologies will enhance the production system through the automation and optimization of production flow convergence, enabling efficiencies and improvements among the suppliers, manufacturers, and consumers in the COVID-19 situation. Originality/value: The study summarizes the impact of the COVID-19 on supply chains and shows the potential of Industry 4.0 technologies to lessen the impact on manufacturing supply chains. This is valuable information for policymakers and practitioners so that they can get insights and take necessary actions.", "keywords": [ "Industry 4.0", "emerging technologies", "supply chain", "COVID 19", "Systematic Literature Review" ], "content": "Introduction\n\nThe COVID-19 pandemic has already had a crucial impact on human health as well as countries’ economies. Supply chains in various industries have been under tremendous pressure to avoid considerable disruptions in their operations.3 COVID-19 has also affected every member of the supply chain process.56,58 The failure of many nations and businesses to deal with the COVID-19 pandemic is attributable to their supply chains and their inability to deliver products and services.63 Supply chain issues, those associated with sourcing techniques, have created substantial disruptions in various supply chains. Lack of risk management, adoption of the single-sourcing strategy, and supplier delivery delays are examples.64 These distractions have generated numerous lessons in understanding supply chain management, advising both researchers and practitioners to reconsider how supply chain strategies should address new disruptive threats.3 In this regard, the role of Industry 4.0 technologies in supply chain management and their revival during COVID-19 is explored in this article.\n\nIndustry 4.0 fosters decentralized manufacturing systems enabled by technological innovations.65 The concept offers a business atmosphere that integrates humans, machines, equipment, and operational processes through Cyber-Physical Systems and the Internet.66 Industry 4.0 integrates its emerging technologies into the entire organizational setting, facilitates automated and dynamic production systems,67 significantly improves the quality of products and services by digitizing the operational activities.68 However, the COVID-19 pandemic emerged when the supply chains had been called upon to transform and adapt the dynamics of Industry 4.0. Incorporating Industry 4.0 technologies has become a strategic imperative for supply chains to improve their competitiveness in the market.1,4 These technologies play a significant role in optimizing the performance of supply chain operations for better results.6 Many academics believe that Industry 4.0 technologies, such as the Internet of Things,5,9,37 big data,14,15,21,38 cloud computing,22,23 additive manufacturing,48,60 and blockchain,17,19,20 can encourage supply chains in times of crisis, and they call for more research in this area.43,45,69 Previous studies on epidemics did not address the employment of emerging technologies in the recovery process61 as well as the impact on commercial supply chains.70 Consequently, it is unclear how a supply chain can utilise technologies to increase flexibility and response time.58 This requires a holistic approach.71 Furthermore, existing literature lacks a comprehensive review on the role of new technologies in enabling supply chains, especially in emergencies such as the COVID-19 pandemic.3,58 Therefore, Chowdhury, Paul, Kaisar and Moktadir58 and Frederico3 suggested looking into the role of emerging technologies of Industry 4.0 in regulating the effects of COVID-19. The current study has conducted a systematic literature review to close this gap. Also, the study assesses the overall role of Industry 4.0 technologies in developing a holistic supply chain framework and focuses on the potential applications of the emerging technologies to address pandemic-related supply chain problems.\n\nHence a comprehensive literature review was conducted on Industry 4.0 technologies, supply chain, and COVID-19 for exploratory analysis and a deeper understanding to answer the following research queries:\n\nQ1. What are the most influential technologies of Industry 4.0 for creating more responsive and resilient supply chains in case of emergencies, such as the COVID-19 outbreak?\n\nQ2. How can the technologies of Industry 4.0 enable supply chains to handle the effects of the COVID-19 outbreak and enhance the responsiveness of the supply chains?\n\n\nMethods\n\nThe study employs systemic literature review (SLR) methodology to get a thorough insight into the relevance of Industry 4.0 technologies in the supply chain during COVID-19.\n\nResearchers have recommended SLR as a comprehensive literature review framework.72 An overview of the SLR process73 followed in this study is shown in Table 1.\n\nTo create a repeatable and impartial search method, the researchers only referred to the most relevant publications connected to the topic. Figure 1 illustrates the three categorical keywords used by the authors to find the most relevant publications. The study adopted the “Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA)” framework developed by Moher74 and the flowchart is visualized in Figure 2. The drafting process was utilised to extract the most relevant articles on the effect of COVID-19 on supply chains and the potential of the emerging technologies to resuscitate supply chains, as stated in the PRISMA standards.\n\n\nResults\n\nFifty-three articles were included for the descriptive and thematic analysis. The reviewed publications showed that the emerging technologies play a significant role in rescuing the disrupted supply chains during the COVID-19 pandemic. The following sub-sections illustrate the descriptive analysis of the publications.\n\nThe descriptive findings of 53 articles are shown in the frequency analysis. Figure 3 depicts a high-level representation of the results. Out of the 53 papers, 48 journals provide 90% of the articles, two conference papers and two book chapters account for 4% each of the total publications, and 2% are the culminating articles.\n\nThe year-wise distribution of the articles is shown in Figure 4. The publication trend demonstrates an impressive growth in the literature, indicating that the topic is well recognised among academics.\n\nFigure 5 presents the distribution of publications among the top seven journals. Sustainability (MDPI) tops with four articles, and benchmarking followed with three articles.\n\nThe contributions of different publishers are shown in Figure 6. Emerald has the most publications with fourteen papers, followed by MDPI and Elsevier with thirteen and nine articles, respectively. This indicates that the concept of Industry 4.0 and supply chains is widely covered.\n\nThe research method-wise distribution of the chosen 53 articles is shown in Figure 7. Empirical methodology tops with 15 papers, followed by general reviews and systematic literature reviews comprising 14 and 12 articles, respectively.\n\nFigure 8 demonstrates the top-cited articles, and the publication by Hofmann, Sternberg, Chen, Pflaum and Prockl24 leads the list with 1035 citations, followed by Queiroz and Wamba44 and Ardito, Petruzzelli, Panniello and Garavelli4 with the second and third highest number of citations, 250 and 196, respectively.\n\nThe list of high-contributing authors is shown in Figure 9. Maciel M. Queiroz tops the list with three publications, followed by Hofmann, Erik; Pereira, Susana Carla Farias; and Sunil, Luthra and Ivanov, Dmitry with two publications each.\n\nFigure 10 shows that India tops the list with ten articles, followed by Australia and Brazil with four and three papers, respectively, then the USA, China and Spain, each with two articles.\n\nFigure 11 illustrates the sector-wise distribution, and the manufacturing supply chains accounts for 29 articles out of 53. Medical & pharmaceuticals, courier, and agri-food supply chains are reflected in each of the three articles.\n\n\nDiscussion\n\nSupply chains have been significantly disrupted during COVID-19, and the fundamental concerns addressed primarily are demand instability and supply shocks.57,58,61,63,64,75–80 COVID-19 not only created havoc in demand and supply but also altered the spending patterns of consumers80,82 and led to inflation.76,79,83 The pandemic also caused reduced sales75,76,84,85 and business shutdowns61,78,84 with huge economic losses in various industries like cars, tourism, and transportation.58,82 This resulted in a shortage of workers78,85 and raw materials63,64,78 and a simultaneous massive failure of production capacity.75,84 This section discusses the recovery strategy for the disrupted supply chains during the COVID-19 pandemic by deploying emerging technologies. The role of these technologies in reviving the supply chains is also underlined.\n\nWith Industry 4.0 technologies, the automated and digital supply chain can be the best solutions for recovering the disrupted supply chains during the COVID-19 pandemic. The role and function of disruptive technologies are essential.3–5\n\nThe literature review highlights the emerging technologies that may streamline supply chain resilience, resulting in increased robustness during an emergency or an unexpected and dynamic catastrophe. The results are summarized in Table 2.\n\nThe manufacturers and producers may adopt and execute emerging technologies such as the Internet of Things (IoT), big data, cloud computing, additive manufacturing, and blockchain to continue, rescue, and recover their supply chains that are affected by the pandemic. Management should dedicate more effort to an automated supply chain developed by the emerging technologies.\n\n\nThe role of the emerging technologies in supply chains\n\nCOVID-19 emphasises the whole production process and the structural factors that link Industry 4.0 technology, supply chains, and the COVID-19 pandemic. In this section, these factors are examined based on the reviews. Table 3 illustrates the role of the emerging technologies in reviving supply chains that favour long-term supply chain performance. The key approaches of the supply chains include real-time information and transparency to manage customer demand effectively,1,4 improved interaction with suppliers and vendors,10,52 and optimising the supply chain to satisfy the needs of the company.40,53 A supply network integrated with emerging technologies enables companies to build faster, flexible, accurate, and efficient supply chains.7 These approaches have a significant influence on supply chain resilience, leading to increased robustness in the face of an emergency or abrupt and large-scale calamities.22 These technologies will improve industrial processes across the horizontal value chain, including engineering, material utilisation, supply chain, and product life cycle management,33,60,86 along with opportunities such as improvements in operations, energy conservation, and logistic support.17,19,87 Productive competency,23,48 waste reduction,1,17 inventory management,19,25 information sharing with supply chain members,54,55 tracking and tracing warehouse inventory,29,88 and logistics information60 are guaranteed by Industry 4.0 technologies.1,21,22 Via data sharing, these technologies facilitate a decrease in local and international bulk cargo transit, delivery mistakes, and needless waiting periods, as well as the prevention of products being damaged.23,48 The review indicates that Industry 4.0 technologies contribute to improving operations management4,5,7 and manufacturing processes creating customized products.89\n\n\nLimitations and future studies\n\nThe authors note several limitations in the study. First, the findings are derived considering English language-publications only, and those written in other languages are excluded. Future research may provide additional insights by reviewing the literature written in other languages. Second, the authors focus on the literature only in the context of Industry 4.0. Thus, the holistic view of Industry 4.0 has not been evaluated in this study. Furthermore, the study reviewed the role of the five major technologies such as IoT, big data, cloud computing, additive manufacturing, and blockchain and discussed how these technologies could be employed to revive the supply chains during emergencies. Future studies may include other emerging technologies such as artificial intelligence, robotics, augmented reality, and simulation/digital twins to get a broader range of findings. In spite of these constraints, the current study adds to the identification of significant technologies and their roles in supply chain management in the area of Industry 4.0.\n\n\nConclusion\n\nRecent studies have emphasized the impact of individual technologies on the supply chain, such as IoT, big data, cloud computing, additive manufacturing, and blockchain, and how these technologies support companies in achieving competitive advantage. However, comparatively few studies have explored the influence of these technologies concurrently, particularly during an unexpected situation. The present study is based on these gaps and responds to the research questions using a systematic literature review. In answering the first research question, the study confirmed that most publications highlight IoT, big data, cloud computing, additive manufacturing, and blockchain (Table 2) that may assist in building resilient and robust supply chains, even in the COVID-19 era. Regarding the second research question, the literature indicates that the roles and functions (Table 3) played by these technologies lead to establishing integrated, flexible, robust, responsive, efficient, and competent supply chains. The study also reveals unexplored features of supply chains. Therefore, highlighting a discussion on implementing Industry 4.0 technologies in supply chain studies offers an interesting future research topic.\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_The Implications of Industry 4.0 on Supply Chains Amid the Covid 19 Pandemic – A Systematic Literature Re.docx, https://doi.org/10.6084/m9.figshare.16602356.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgement\n\nThe authors would like to acknowledge Multimedia University for supporting the research.\n\n\nReferences\n\nAcioli C, Scavarda A, Reis A: Applying Industry 4.0 technologies in the COVID–19 sustainable chains. Int J Productivity Performance Management. 2021; 70(5): 988–1016. 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Publisher Full Text\n\nTortorella GL, Fettermann D: Implementation of Industry 4.0 and lean production in Brazilian manufacturing companies. Int J Production Res. 2018; 56(8): 2975–2987. Publisher Full Text\n\nFatorachian H, Kazemi H: Impact of Industry 4.0 on supply chain performance. Production Planning & Control. 2021; 32(1): 63–81. Publisher Full Text\n\nEr Kara M, Oktay Fırat SÜ, Ghadge A: A data mining-based framework for supply chain risk management. Computers Industrial Eng. 2020; 139: 105570. Publisher Full Text\n\nIvanov D, Das A: Coronavirus (COVID-19/SARS-CoV-2) and supply chain resilience: A research note. Int J Integrated Supply Management. 2020; 13(1): 90–102. Publisher Full Text\n\nArsovski S, Arsovski Z, Stefanović M, et al.: Organisational resilience in a cloud-based enterprise in a supply chain: a challenge for innovative SMEs. Int J Computer Integrated Manufacturing. 2017; 30(4-5): 409–419. 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Publisher Full Text\n\nHobbs JE: Food supply chains during the COVID-19 pandemic. Can J Agricultural Economics/Revue canadienne d'agroeconomie. 2020; 68(2): 171–176. Publisher Full Text\n\nKumar A, Luthra S, Mangla SK, et al.: COVID-19 impact on sustainable production and operations management. Sustainable Operations Computers. 2020; 1: 1–7. Publisher Full Text\n\nWang Y, Wang J, Wang X: COVID-19, supply chain disruption and China’s hog market: a dynamic analysis. China Agricultural Economic Rev. 2020; 12(3): 427–443. Publisher Full Text\n\nde Paulo Farias D, de Araújo FF: Will COVID-19 affect food supply in distribution centers of Brazilian regions affected by the pandemic?. Trends Food Science Technol. 2020; 103: 361–366. Publisher Full Text\n\nBarman A, Das R, De PK: Impact of COVID-19 in food supply chain: Disruptions and recovery strategy. Curr Res Behav Sci. 2021; 2: 100017. Publisher Full Text\n\nHobbs JE: Food supply chain resilience and the COVID-19 pandemic: What have we learned?. Can J Agricultural Economics/Revue canadienne d'agroeconomie. 2021. Publisher Full Text\n\nGovindan K, Mina H, Alavi B: A decision support system for demand management in healthcare supply chains considering the epidemic outbreaks: A case study of coronavirus disease 2019 (COVID-19). Transportation Res Part E: Logistics Transportation Review. 2020; 138: 101967. Publisher Full Text\n\nBelhadi A, Kamble S, Jabbour CJC, et al.: Manufacturing and service supply chain resilience to the COVID-19 outbreak: Lessons learned from the automobile and airline industries. Technological Forecasting Social Change. 2021; 163: 120447–120447. Publisher Full Text\n\nAgrawal S, Jamwal A, Gupta S: Effect of COVID-19 on the Indian Economy and Supply Chain. Preprints. 2020. Publisher Full Text\n\nJayashree S, Reza MNH, Mohiuddin M: Impact of Cleaner Production and Environmental Management Systems on Sustainability: The Moderating Role of Industry 4.0. IOP Conference Series: Earth and Environmental Science. 2021; 795(1): 012013. Publisher Full Text\n\nJayashree S, Malarvizhi C, Reza MNH: The Challenges and Opportunities of Industry 4.0-A Review. Asia Proc Social Sci. 2020; 5(2): 173–178. Publisher Full Text\n\nQueiroz MM, Ivanov D, Dolgui A, et al.: Impacts of epidemic outbreaks on supply chains: mapping a research agenda amid the COVID-19 pandemic through a structured literature review. Ann Operations Res. 2020; 1–38. Publisher Full Text\n\nBelinski R, Peixe AMM, Frederico GF, et al.: Organizational learning and Industry 4.0: findings from a systematic literature review and research agenda. Benchmarking: Int J. 2020; 27(8): 2435–2457. Publisher Full Text" }	[ { "id": "96185", "date": "12 Oct 2021", "name": "Guilherme Brittes Benitez", "expertise": [ "Reviewer Expertise Industry 4.0 and supply chain." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the manuscript entitled “The implications of Industry 4.0 on supply chains amid the COVID-19 pandemic: a systematic review”. The subject is timely and relevant and the authors did a nice job in their methodological procedures. However, further improvements are necessary for this version to be suitable for indexing.\nAbstract:\n“Purpose: This research examines the effects of the epidemic on supply chains and how these effects are reduced through Industry 4.0 technology.” <- Industry 4.0 technologies\n“Findings: This study discusses the impact of COVID-19 on the supply chain and how the emerging technologies of Industry 4.0 can help manufacturers to ease the impact.” <- this is not a result, this is justificative of your research\n“These technologies will enhance the production system through the automation and optimization of…” <- “will” is quite odd here. Are you concluding this from the SLR or is this the main finding from your research?\nOriginality/value is quite weak. It looks like more a justificative than the real contribution from your work.\nIntroduction:\nOverall, the introduction section is well written and the flow of information sounds good when reading the text. Congrats!\n“In this regard, the role of Industry 4.0 technologies in supply chain management and their revival during COVID-19 is explored in this article.” <- What do you mean by saying “their revival”? It seems out of context here.\nResearch method:\nThis section needs improvements. It is too superficial and the authors try to give all the explanations in Table 1 and Figures 1 and 2. This is not sufficient to explain all your methodological procedures. Please, better explain your steps when doing this SLR.\nWhy did you consider 2016, 2017, and 2018 in your research given the covid-19 pandemic started in December 2019? From the perspective proposed in the manuscript, you have at least 8 articles you should not include in your analysis.\nYou have to better justify why you considered before and pre-pandemic periods.\nResults:\nThe bibliometric analysis is good. I enjoyed seeing how the authors give special attention to this point.\nI believe the authors should explain all images in this section. Despite some images being easy to follow a critical analysis would enrich and benefit the manuscript.\nDiscussions:\nI believe the authors mixed the results and discussions sections. Tables 2 and 3 certainly must be in the Results section and therefore they should be discussed in the discussions section.\nI cannot see the link between Industry 4.0 technologies and each category and its respective advantages. If you create a new column explaining which technologies are related it certainly would help readers. Moreover, you should better present (discuss) these points in your results section.\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": "7643", "date": "31 Jan 2022", "name": "Mohammad Nurul Hassan Reza", "role": "Author Response", "response": "We would like to thank you for all the valuable comments. We believe that the revised manuscript has addressed all the concerns. Below are the response to the comments: Reviewer's comments: “Purpose: This research examines the effects of the epidemic on supply chains and how these effects are reduced through Industry 4.0 technology.” <- Industry 4.0 technologies Author Response to Comments Thank you very much for addressing the typo. We have already done the correction. Reviewer's comments: “Findings: This study discusses the impact of COVID-19 on the supply chain and how the emerging technologies of Industry 4.0 can help manufacturers to ease the impact.” <- this is not a result, this is justificative of your research. Author Response to Comments Thank you very much for pointing this out. The findings are revised as follows: Findings: The findings of the study showed that the majority of the articles emphasized the digitalization of supply chain management, acknowledging the fundamentals, applications, and prospects, revealing the drivers and challenges of Industry 4.0 technologies to manage disruptions. Most of the authors identified IoT, big data, cloud computing, additive manufacturing, and blockchain to maintain the supply chain resilience. Reviewer's comments: “These technologies will enhance the production system through the automation and optimization of…” <- “will” is quite odd here. Are you concluding this from the SLR or is this the main finding from your research? Author Response to Comments Thank you very much for suggesting. We have revised the statement accordingly. Reviewer's comments: Originality/value is quite weak. It looks like more a justificative than the real contribution from your work. Author Response to Comments We have addressed this and revised it as follows: Originality/value: Existing literature on epidemics lacks the basics and practices of utilizing Industry 4.0 technologies in the supply chain recovery process. To fill this research gap, the study summarizes the potential of Industry 4.0 technologies to lessen supply chain disruptions caused by COVID-19. The study findings are valuable for policymakers and practitioners and contribute to supply chain management studies. Reviewer's comments: “In this regard, the role of Industry 4.0 technologies in supply chain management and their revival during COVID-19 is explored in this article.” <- What do you mean by saying “their revival”? It seems out of context here. Author Response to Comments Thank you very much for noticing this. We have already amended the statement. Reviewer's comments: This section needs improvements. It is too superficial and the authors try to give all the explanations in Table 1 and Figures 1 and 2. This is not sufficient to explain all your methodological procedures. Please, better explain your steps when doing this SLR. Author Response to Comments Thank you very much for your suggestion. Due to the word limitation, we planned to use tables and figures to provide some information. However, as the journal advised elaborating relevant sections to respond to the reviewers’ comments, all of the methodological procedures are further explained, including the steps of the articles selection process. Reviewer's comments: Why did you consider 2016, 2017, and 2018 in your research given the covid-19 pandemic started in December 2019? From the perspective proposed in the manuscript, you have at least 8 articles you should not include in your analysis. You have to better justify why you considered before and pre-pandemic periods. Author Response to Comments Thank you very much for your suggestion. Initially, we included some papers published before 2019 as the focus of these papers was on Industry 4.0 relating to supply chain disruption. However, to avoid confusion, we have removed the articles published before 2019, and all the figures, tables, and discussions have been revised accordingly. Reviewer's comments: The bibliometric analysis is good. I enjoyed seeing how the authors give special attention to this point. I believe the authors should explain all images in this section. Despite some images being easy to follow a critical analysis would enrich and benefit the manuscript. Author Response to Comments Thank you very much for your suggestion. Due to the word limitation, we tried to briefly explain the graphs/images. However, as the journal advised to elaborate relevant sections suggested by the reviewers, further explanation is added for each graph/image in the bibliometric analysis. Reviewer's comments: I believe the authors mixed the results and discussions sections. Tables 2 and 3 certainly must be in the Results section and therefore they should be discussed in the discussions section. Author Response to Comments Thank you very much for addressing the issue. Tables 2 and 3 are updated and inserted in the results section. The core technologies of Industry 4.0 are identified in Table 2, followed by Table 3, demonstrating the advantages of employing these technologies in supply chains during disruptions. Reviewer's comments: I cannot see the link between Industry 4.0 technologies and each category and its respective advantages. If you create a new column explaining which technologies are related it certainly would help readers. Moreover, you should better present (discuss) these points in your results section. Author Response to Comments Thank you very much for the suggestion. We have added Table 3 (Advantages of employing emerging technologies in supply chains.) and Table 4 (Summary of reviewed literature) to address the issue. Furthermore, the categorical analysis is done where the prospects of Industry 4.0 technologies in supply chains are identified and classified into clusters and sub-clusters following a thorough interpretation of all the selected articles." } ] }, { "id": "96184", "date": "21 Oct 2021", "name": "Lorenzo Ardito", "expertise": [ "Reviewer Expertise Innovation", "digital transformation" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is more a bibliometric analysis than a systematic review. A systematic review paper includes a discussion of the articles selected. This article mainly provide some descriptive statistics.\nFrom these statistics, it is hard to see answers about the two questions. That is, where can I see the \"most influential technologies of Industry 4.0 for creating more responsive and resilient supply chains in case of emergencies, such as the COVID-19 outbreak\"? Where did you explain \"how can the technologies of Industry 4.0 enable supply chains to handle the effects of the COVID-19 outbreak and enhance the responsiveness of the supply chains?\" The article must answer these questions delving into the outcomes of the included papers.\nAlso, the covid pandemic started at the end of 2019. Why are articles published earlier included?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [ { "c_id": "7644", "date": "31 Jan 2022", "name": "Mohammad Nurul Hassan Reza", "role": "Author Response", "response": "Reviewer's comments: The study is more a bibliometric analysis than a systematic review. A systematic review paper includes a discussion of the articles selected. This article mainly provide some descriptive statistics. From these statistics, it is hard to see answers about the two questions. That is, where can I see the \"most influential technologies of Industry 4.0 for creating more responsive and resilient supply chains in case of emergencies, such as the COVID-19 outbreak\"? Where did you explain \"how can the technologies of Industry 4.0 enable supply chains to handle the effects of the COVID-19 outbreak and enhance the responsiveness of the supply chains?\" The article must answer these questions delving into the outcomes of the included papers. Author Response to Comments Thank you very much for your comments. Initially, we were bound to prepare a short SLR due to the limitation of 2500 words. However, the journal has advised elaborating all the relevant sections to address the reviewers’ comments. The revised version addressed all the concerns raised, and further explanation is added in the appropriate sections. In the revised version of the manuscript, the discussion section is now improved with two separate tables. Table 3 illustrates the reviewed literature where the potential advantages of Industry 4.0 technologies in supply chain resilience are identified. The reviewed articles are summarized in Table 4, including the technologies of Industry 4.0 that were the main focus of each study. The research area, methodological approach, and findings of each of the studies are also presented. Additionally, categorical analysis is done with a framework illustrating the findings of the study and answering the research questions. In this section, the key enabling technologies of Industry 4.0 in establishing resilient supply chains and the potential advantages (clusters) have been identified. The discussion on the sub-clusters of each cluster is also added, answering the second research question. Reviewer's comments: Also, the COVID pandemic started at the end of 2019. Why are articles published earlier included? Author Response to Comments Thank you very much for your suggestion. Initially, we included some papers published before 2019 as the focus of these papers was on Industry 4.0 relating to supply chain disruption. However, to avoid confusion, we have removed the articles published before 2019, and all the figures, tables, and discussions have been revised accordingly. Yet, the authors retained at least three articles focusing on general supply chain disruptions rather than COVID-19, published in 2019. Thank you very much." } ] }, { "id": "96186", "date": "15 Nov 2021", "name": "Chetna Chauhan", "expertise": [ "Reviewer Expertise Industry 4.0", "Circular economy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 needs to address some important shortcomings before it can be accepted for indexing.\nIn Introduction the authors need to clearly state the rationale behind this study, i.e., why do they think that technologies can be useful in the covid time.\nThe method section also requires details. See papers on SLR methodology.\nThe authors review a huge set of 53 articles, but the discussion is very brief. The authors need to elaborate their discussion by highlighting the key themes and sub-themes. Include a section on gaps in the existing studies and future research directions, which is based on your review (see food waste papers by this reviewer: Chauhan et al. 2021a1, Chauhan et al. 2021b2).\n\nLanguage check is a must. There are typos and errors, for example, see figure 5 \"nmber,\" etc.\nBest of luck.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "7645", "date": "31 Jan 2022", "name": "Mohammad Nurul Hassan Reza", "role": "Author Response", "response": "We would like to express our cordial thanks and gratitude to you for reviewing the manuscript. We believe that the revised version has addressed all the issues. Below are the response to the comments: Reviewer's comments: In Introduction the authors need to clearly state the rationale behind this study, i.e., why do they think that technologies can be useful in the covid time. Author Response to Comments Thank you very much for your suggestion. The introduction section is further explained to address this issue. Reviewer's comments: The method section also requires details. See papers on SLR methodology. Author Response to Comments Thank you very much for pointing this out. Due to the word limitation, we planned to make a short SLR using tables and figures to provide the relevant information. However, as the journal advised elaborating relevant sections to respond to the reviewers’ comments, all of the methodological procedures are further explained, including the steps of the article selection process. Reviewer's comments: The authors review a huge set of 53 articles, but the discussion is very brief. The authors need to elaborate their discussion by highlighting the key themes and sub-themes. Author Response to Comments Thank you very much for your comments. Initially, we were bound to prepare a short SLR due to the limitation of 2500 words. However, the journal has advised elaborating all the relevant sections to address the reviewers’ comments. In the revised version of the manuscript, the discussion section is now improved with two separate tables. Table 3 illustrates the reviewed literature where the potential advantages of Industry 4.0 technologies in supply chain resilience are identified. The reviewed articles are summarized in Table 4, including the technologies of Industry 4.0 that were the main focus of each study. The research area, methodological approach, and findings of each of the studies are also presented. Additionally, categorical analysis is done with a framework illustrating the findings of the study and discussing the themes and sub-themes. In this section, the key enabling technologies of Industry 4.0 in establishing resilient supply chains and the potential advantages (clusters/themes) have been identified. The discussion on the sub-cluster/sub-theme of each cluster/theme is also added, answering the research questions of the study. Reviewer's comments: Include a section on gaps in the existing studies and future research directions, which is based on your review. Author Response to Comments Thank you very much for the suggestion. A section illustrating the research gaps identified in the reviewed literature and future directions is added." } ] } ]	1	https://f1000research.com/articles/10-1008
https://f1000research.com/articles/9-1379/v1	27 Nov 20	{ "type": "Systematic Review", "title": "Applications of GIS and geospatial analyses in COVID-19 research: A systematic review", "authors": [ "Rakibul Ahasan", "Md. Shaharier Alam", "Torit Chakraborty", "Md. Mahbub Hossain", "Md. Shaharier Alam", "Torit Chakraborty", "Md. Mahbub Hossain" ], "abstract": "Background: Geographic information science (GIS) has established itself as a distinct domain and incredibly useful whenever the research is related to geography, space, and other spatio-temporal dimensions. However, the scientific landscape on the integration of GIS in COVID-related studies is largely unknown. In this systematic review, we assessed the current evidence on the implementation of GIS and other geospatial tools in the COVID-19 pandemic. Methods: We systematically retrieved and reviewed 79 research articles that either directly used GIS or other geospatial tools as part of their analysis. We grouped the identified papers under six broader thematic groups based on the objectives and research questions of the study- environmental, socio-economic, and cultural, public health, spatial transmission, computer-aided modeling, and data mining. Results: The interdisciplinary nature of how geographic and spatial analysis was used in COVID-19 research was notable among the reviewed papers. Although GIS has substantial potential in planning to slow down the spread, surveillance, contact tracing, and identify the trends and hotspots of breakdowns, it was not employed as much as it could have been. This review not only provided an overarching view on how GIS has been used in COVID-19 research so far but also concluded that this geospatial analysis and technologies could be used in future public health emergencies along with statistical and other socio-economic modeling techniques. Our systematic review also provides how both scientific communities and policymakers could leverage GIS to extract useful information to make an informed decision in the future. Conclusions: Despite the limited applications of GIS in identifying the nature and spatio-temporal pattern of this raging pandemic, there are opportunities to utilize these techniques in handling the pandemic. The use of spatial analysis and GIS could significantly improve how we understand the pandemic as well as address the underserviced demographic groups and communities.", "keywords": [ "GIS", "Coronavirus", "COVID-19", "Spatial analysis", "Systematic review", "Evidence-based practice" ], "content": "Introduction\n\nCOVID-19 has taken the world within a blink of an eye with a rapidly increasing confirmed cases and case-fatalities around the world1. After originating in the Hubei province of China in late December 2019, the World Health Organization (WHO) termed it an epidemic on January 29 20202, named it COVID-19 on February 11, and declared it as a pandemic on March 113. Although the first reported case was in China, and it was the epicenter of the pandemic, the virus has mutated and changed transmission pattern several times since then. Lately, the United States, parts of Europe, and countries in the Global South had been reporting the highest number of cases with a rapid increase in both confirmed cases and fatalities1.\n\nThe declaration of COVID-19 as a pandemic and subsequent lockdown at various levels, from the local city level to the country level, has a much broader impact on our surrounding environment compared to what we usually observe. Despite the availability of data, studies examining the impacts of this ongoing pandemic and enforced lockdowns using different geospatial analysis techniques is not substantial. However, the application of spatial analysis tools, techniques, and geographic information system (GIS) platforms provide the scientific community and the practitioners a wide range of benefits. These benefits include more straightforward and understandable visualization, real-time tracking of confirmed and reported case numbers4, contact tracing, spread direction, and also, to identify the hotspots to limit the dispersion and community spread5,6. The application of GIS in public-health related issues is not something introduced during this pandemic. It was used by numerous studies in the past, long before the computerized GIS software was born in the mid-1960s7. Since then, GIS was extensively used in analyzing, visualizing, and detecting patterns of disease. A recent review found that among the included 869 studies, one-fourth of the studies used GIS techniques for mapping, especially infectious disease mapping8.\n\nDifferent GIS software and methods have been implemented and widely accepted to prevent the transmission by imposing lockdowns and contact tracing. The best example of GIS application during this pandemic is the web-based near-real-time COVID dashboard created by the Johns Hopkins University4,9,10. Later the WHO and different local and regional governing bodies also followed the same direction11. The online dashboards have been a critical source of information during this pandemic. Although at the beginning the studies implementing or using GIS methods were more focused on visualizing or contact tracing, later, they moved on spatial analysis incorporating social, economic, environmental, and more sophisticated analytical tools as more data started to become available.\n\nThere have been attempts to review the studies regarding the application of geospatial analysis in COVID-19 related studies. Pardo et al. (2020) reviewed the studies that were focused on understanding the distribution patterns of the pandemic and identified such applications in six thematic groups12. Similar attempts were made by Kamel Boulous and Geraghty (2020) to review the web-based use of GIS technologies6. However, none of these approaches followed a systematic approach in selecting the articles, and the reviews were mostly incomprehensive.\n\nSystematic reviews provide an organized, replicable, and methodologically synthesized landscape of evidence that may inform policymaking and practice. During this pandemic, the scarcity of evidence remains a major challenge for public policymaking, which necessitates a careful assessment of the growing body of literature on GIS and geospatial analyses. Also, systematically evaluated evidence is critical for the advancement of science as further primary studies or research syntheses can be informed by the findings of a systematic review. We acknowledged this knowledge gap and conceptualized this review to advance science and practice related to GIS and a wide range of geospatial techniques that are being used in this pandemic. The objective of this study is to conduct a systematic review of the implementation of GIS and other geospatial tools and technologies in COVID-19 related studies. We highlighted the works that used geospatial techniques as part of their analytical method and tried to provide pointers on how these techniques can be better used in times of future public health emergencies.\n\n\nMethodology\n\nThis systematic review was conducted using the reporting guidelines as stated in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement13. A protocol was prepared before conducting this review that was not registered with PROSPERO or any other organization. This protocol was uniformly followed by the reviewers at each stage of the review, which is available upon request. The data for this systematic review were retrieved from MEDLINE and Web of Science. Both databases have their own competitive advantages that provide a broader coverage of scholarly articles. MEDLINE is considered as the largest bibliographic databases for health sciences, whereas Web of Science provides access to journals from multiple scholarly disciplines. We used the following steps to identify relevant literature for this review. First, we used the following search query in each database: (“COVID-19” OR “2019-nCoV” OR “2019 coronavirus” OR “2019 novel coronavirus” OR “novel coronavirus” OR “SARS-CoV-2”) to identify COVID-19 related studies. Further, we used another search query to retrieve GIS-related studies as following: (“GIS” OR “ArcGIS” OR \"Geographic information systems\" OR \"Geographic mapping\" OR \"Spatial analysis\" OR \"Geospatial analysis\"). At the next step, we combined both these queries with “AND” operator to identify literature that is likely to contain studies referring to both these topics. Furthermore, as COVID-19-related literature is evolving rapidly, we also searched the Google Scholar database to identify studies that may align with the objective of this review. Also, we performed a reference searching and contacted subject matter experts for additional studies beyond the scope of the databases, if there were any. We limited the timeline for literature searching between 2019 and 2020 considering the origin of the outbreak in late 2019. The search was first conducted on May 7, 2020 and updated on June 28, 2020.\n\nArticles were considered eligible for this review if they were a) published in English language, b) available as peer-reviewed journal articles, c) the primary focus of the paper was on any aspect of the COVID-19 pandemic, d) demonstrated the applications of GIS or geospatial analyses. Any article that did not comply any of these criteria were excluded from this review. For example, non-English studies, articles that were not peer-reviewed (e.g., letters, editorials, comments), studies that did not focus on COVID-19, or did not use GIS or geospatial techniques (conceptual papers without providing any findings) were considered ineligible for inclusion.\n\nAll citations retrieved through database searching were imported in Endnote reference manager software for curating the collective bibliography. Further, this library was exported to Rayyan QCRI, a cloud-based software for citations screening and assessment. Two authors independently assessed each citation against the eligibility criteria stated above. At the end of the process, a third reviewer was consulted to review the conflicts and a consensus on inclusion or exclusion was made upon discussion. The full texts of the primarily selected articles were reviewed and reviewed by all authors.\n\nFurther, articles fulfilling all criteria for this review were retained and data were extracted using a predesigned extraction sheet in Microsoft Excel on the following variables:\n\na) publication details,\n\nb) study objectives,\n\nc) the sources of data,\n\nd) countries of origin,\n\ne) COVID-19 specific domain presented in the papers,\n\nf) the use of GIS or geospatial analyses, and\n\ng) the research outcomes or key findings of those studies.\n\nThe applications of GIS or geospatial analyses on COVID-19 studies were identified and narratively synthesized as major themes alongside tabulation of the key findings. A narrative synthesis is appropriate where quantitative or qualitative synthesis may not be feasible due to the methodological differences and other measures of heterogeneity across the included studies14. Moreover, quality appraisal or risk of bias assessment were not considered in this review due to a large volume of studies and profound heterogeneity in methodological approaches, data sources, measurements, types of applications adopted in different contexts, and research outcomes in respective studies.\n\n\nResults\n\nThe PRISMA diagram for this review is shown in Figure 1. We found a total of 79 articles which met all the inclusion criteria. A PRISMA checklist enlisting the contents of this systematic review is available on Open Science Framework repository15. A summary of these 79 reviewed articles are listed in Extended data, Table 116. The articles included in the review were published in a diverse group of journals, mostly in public health, urban planning, geography, and interdisciplinary journals. Although COVID-19 is more of a public health and welfare issue, these articles covered from public health issues to planning techniques, environmental concerns, and, most importantly, used geospatial analysis tools and techniques as part of their methods. In total, 32.9% of the articles (n=26) used China and 21.5% the United States (n=17) as their study site, which indicates to the disparity of the geographic coverage. Figure 2 shows the geographic distribution of the study area used in the 79 articles we included in our review. Wuhan, China, first reported COVID-19 cases, and the GIS has the highest number of confirmed cases, which also was reflected in the over-representation of these two countries in these articles. Africa and Europe had the lowest number of articles (n=4). Among these reviewed articles, 15% (n=12) worked on a global or multi-national scale. The global studies are more focused on mobility and how COVID-19 transmitted via airports and other human mobility17–19.\n\n(a) article count per country and geographic distribution of the study sites; (b) number of articles in each continents based on study site along with Global scale studies; (c) number of articles in each thematic groups used in this review; and (d) bubble chart showing the frequency of each software or tool used for spatial analysis.\n\nA wide range of geospatial tools were used in the reviewed articles. Most of these articles used regression analysis (n=18) and correlation analysis (n=11) for statistical analysis. Most prominently used spatial analysis was hotspot analysis using either kernel density function or other density techniques (n=16) followed by the spatial autocorrelation analysis using either global or local Moran’s Index (n=13) and proximity analysis (n=5). All the articles used different visualization techniques to display either the intermediate or the end products. Along with that, suitability analysis (n=3) and sensitivity analysis (n=4) was also used to find either access to hospitals or how the cases are distributed. One of the less frequently implemented but interesting technique was to track transmission patterns using the mobility data20–22. Similarly, remote sensing data was used for air quality and pollution measurement, but not that prominently (n=6). Nighttime imagery and solar radiation data were used by n=5 studies to compare the energy consumption differences between before and after pandemic situation.\n\nESRI ArcGIS is the most used platform (n=36) in these articles, but other open-source platforms like RStudio, where spatial and statistical analysis can be done simultaneously, were often used as well (n=6). Besides RStudio, QGIS (n=4) is another open source software platform that was highly employed by the researchers. Apart from that, a couple of the studies developed their own platforms and one of the studies used GPS. data. Most of these articles used COVID-19 confirmed cases as part of their analysis (n=77). In most cases, researchers used either WHO provided data or JHU provided data for the research purpose. As several spatial analysis was performed based on remotely sensed data, NASA and USGS satellite images were the primary source for those (n=5). Apart from that, the air quality index (AQI) was used in the articles focused on environmental analysis (n=8). As mentioned in the methodology, we did not assess the risk of bias among studies included in this review.\n\nThe rapid diffusion of COVID-19 and data convenience had enforced the global scientific community to work more vigorously on geospatial analysis of this pandemic. These studies had focused on distinct aspects of the pandemic and have different inputs. The articles included in this review was divided into six thematic groups- environment (n=25), socio-economic and cultural (n=13), Population Health Surveillance (n=8), spatial transmission (n=9), computer-aided spatial and statistical analysis and modeling (n=17) and big data, social media and mobile data (n=10).\n\nEnvironment. We found a total of 25 articles that emphasized the use of GIS and spatial analysis on environmental issues related to COVID-1918,20–43. Although these studies had a similar research interest, they differ significantly based on the spatial scale- from global23–27 to regional28,29, national30–38 and small local scale studies39–45. Several works examined the relationship between different meteorological factors and the transmission potential of the virus. They included a diverse set of characteristics in the studies: temperature21,25–27,30,31,33,35–37,39,42,44,46, humidity21,26,27,31,33,35–37,39,44,46, precipitation21,39, daylight hours25,46, solar radiation21,31,35, and wind-speed27,33,35,39. One primary hypothesis of these studies was that these climatic factors and the dispersion of COVID-19 are correlated. In an attempt to examine the hypothesis, statistical and geospatial analysis was used as a key analytical tool25,31,39,44.\n\nStudies analyzing the effects of temperature, humidity, and solar radiation found that an association exists between the transmission of the virus and humidity. These studies claimed that humidity is directly associated with the transmission31, while temperature has no relation to the viruses diffusion35,37. However, another study refuted these findings and claimed both temperature and average daylight hours are influencing the spread of the virus25. Other studies incorporated demographic variables as well as mobility and infections data with these meteorological factors to investigate how they are correlated with the COVID-19 outbreak35. Instead of concentration mapping, they showed the spatial distribution and sensitivity of each factor in the study area map and asserted that population density and human mobility among the provinces are directly influencing the accelerated diffusion of COVID-19 cases.\n\nStatistical and spatial analysis and modeling were used in other studies to identify the association between COVID-19 and climatic factors to understand the spatial distribution pattern of COVID-19 cases26,44 and spatio-temporal prediction of the pandemic for a different period36. These studies concluded that without adequate and effective control measures, there is no evidence of humidity, and summer weather substantially limiting the pandemic growth26. Additionally, Pearson's correlation, generalized additive model, and regression models were also utilized in different studies to understand the influence of climate on the virus transmission. These works widely used GIS for mapping the observed and predicted COVID cases30,33, mean temperature and humidity variation and correlation with COVID cases27. None of these studies found any evidence of slower COVID-19 transmission with the change of temperature and humidity.\n\nDifferent countries around the world have enforced different lockdown strategies since mid-January, even before WHO declared COVID-19 as a pandemic. As a result of these lockdowns, and lowered human and industrial activities, a substantial decline in air pollution was noticed23,24,28,29,34,40,41,43. Several studies had reported a significant reduction in NO2, CO, and SO2 concentration in the environment23,24,28,34,40. Studies reported significant improvement in AQI39 and significant reductions in particulate matter (PM) concentrations41. Similar studies at a global scale reported a significant decrease in NO2 concentration and minor decrease in CO concentration, and aerosol optical depth23.\n\nOther studies identified a global reduction of environmental pollution by up to 30%, human mobility by up to 90%24, and vessel activities by up to 69%29. A study using satellite images to compare the pollutants concentration before, during, and after the Chinese Spring Festival observed that the usual trend of NO2 and SO2 concentrations decrease before the festival and increase afterwards was not noticed in 202034. ArcGIS was extensively used in several studies to quantify the association between AQI and the distribution of COVID-19 cases32,33. These studies claimed that air quality is a core driver of COVID-19 dispersion around the world, and the dispersion enhances in a temperature ranged from 10 to 20°C. ArcGIS was also used to perform sensitivity analysis, and for the calculation of Global Moran's I and LISA to analyze the impact of PM concentration in the air on the fatality rate in China45. The study concluded that the fatality rate is positively correlated with the pollutant concentrations in the air.\n\nSocio-economic and cultural. In response to the coronavirus pandemic, alongside the booming clinical and public health research, social scientists are also retooling existing studies, methodologies, and data to understand the people's behavior responding to the pandemic and its impact on socio-economic and cultural settings around the globe. A total of 12 articles were identified that emphasized on socio-economic and cultural aspects in COVID-19 and used GIS43–54. Researchers used gross domestic product (GDP), demographic and household compositions data, population density, and accessibility data46. Several researchers utilized existing social vulnerability index data and applied geospatial tools and techniques to examine the spatial pattern of COVID-19 emergence across different socio-cultural settings47–49. Studies developed dot density and choropleth maps of COVID-19 cases and reported that population density diseases along with poverty and unemployment rates are the major indicators associated with a higher COVID-19 mortality rate49.\n\nDifferent studies reported association among spatial distribution of the socioeconomic variables and the temporal progression of the pandemic using a regression model based spatio-temporal analysis. They noticed that per capita GIS and public transit access is closely related to COVID-19 incidence46. Using a similar analytical tool, another study estimated the association between virus infection and social vulnerability considering county-level socioeconomic data, demographic composition, disability and minority status, language, and housing and transportation database. The study concluded that the increase of COVID-19 cases are highly associated with minority status and language47. To understand the association between racial inequality and COVID-19 mortality, Kim and Bostwick (2020) utilized principal component analysis (PCA) and hotspot analysis in ArcGIS. The study reported that African American communities are the ones with the highest COVID-19 related deaths in the USA48. Other studies also used ArcGIS and regression analysis to investigate the spatial patterns of the COVID-19 in relation to socio-economic variables. This study used GIS to map the spatial aspects and disparities between metropolitan and nonmetropolitan communities and the regression analyses to test the hypotheses of positive correlations between COVID-19 incidence and mortality rates and socio-economic factors in the GIS50.\n\nProximity and hotspot analysis in GIS has become a widely used geospatial analysis technique in the social research to understand the feasibility of social distancing51, accessibility analysis of specific age group52,53. Gibson and Rush (2020) calculated the distance to each dwelling's nearest neighbors to identify units that are unable to practice social distancing effectively. Dryhurst et al. (2020) developed a global risk perception index (R.P.I.) using a linear regression model and used GIS to plot the mean risk perception of COVID-19 in 10 countries54. Sarkar (2020) used ArcGIS to reclassify the administrative units of Bangladesh based on COVID-19 susceptibility using multicriteria analysis based pairwise comparison55. Cavalcante and Abreu (2020) applied Moran’s I and LISA to identify the type and degree of spatial clustering and scatter plots of socio-economic indicators56. Similarly, Exploratory Spatial Data Analysis (ESDA) technique was used to identify spatial relationships between the density of built heritage resources and Airbnb listings. Based on the calculation and mapping, they concluded that the distribution of Airbnb listings has a certain degree of spatial autocorrelation57.\n\nPopulation health surveillance. From the first case in Wuhan to the global pandemic, an enormous number of public health studies have been conducted to help the policymakers to understand how best to manage the current and future public health responses. A total of nine articles were identified that emphasized public health issues and used GIS50,55–62. Multiple studies developed a multicriteria decision making index to assess the risk and resilience of the existing healthcare system. Requia et al. (2020) employed GIS techniques to construct a geodatabase comprising land use, income, population, health condition, number of hospital beds, and staff at the municipality scale. They predicted a deficit of 17 beds in Brazilian municipalities55. Similarly, Jovanović et al. (2020) developed a global index comprising of 57 indicators using ArcGIS-based network analysis for hospital accessibility and resilience mapping58,59.\n\nGIS was used to find out whether there is any association between orthopedic surgeons’ age and COVID-19 confirmed cases60. The study reported a spatial relationship between the number of cases and number of surgeons in a state. In a similar study, Ruthberg et al. (2020) studied the potential risk of otolaryngologists above age 60 due to COVID-19, where they used a heat map to show the state-wise potential risk zone using QGIS. The study indicated that New York, New Jersey, Massachusetts, and Michigan were the riskiest zone according to the ratio of the number of confirmed cases to the number of total ENT's over 6061. Kuupiel et al. (2020) calculated the distance from the primary healthcare (PHC) clinic as well as to the nearest health facility in ArcGIS to measure geographical accessibility (in terms of distance and time) to COVID-19 specialized hospital facilities in Ghana. The analysis reported that the current mean travel time (more than an hour) and distance (more than 35 kilometers) to PHC is significantly higher than the globally accepted standards62. Similar accessibility analysis was done in Australia using proximity analysis and hotspot analysis to measure travel time to the closest hospital facility for aged population resulted in a similar outcome as well53.\n\nAhmadi et al. (2020) developed an epidemic prediction model for spatial-temporal analysis to predict and estimate the number of patients and deaths at the end of pandemic (infected, cured, and dead cases). The study predicted that approximately 7900 and 4620 deaths would occur in Iran from May 13 to June 1, 2020, respectively, and then the curve will flatten63. To analyze the epidemiology of COVID-19, studies also utilized georeferencing. These studies first geocoded all COVID-19 cases and then joined them to the county layers by administrative codes in ArcGIS and afterwards, applied LISA. This study reported that the spatial clustering is not random and shows significant spatial heterogeneity in China64. A wastewater-based epidemiology (WBE) tool was proposed as a surveillance tool to monitor the COVID-19 pandemic65. The study used multiple variables to run the GIS-based hydraulic model and network analysis using the SWMM modeling environment and ArcGIS. The result effectively served as a justification to use WBE as a rapid and efficient tool to track COVID-19, which the authors claimed could be used with clinical testing to save billions of dollars65.\n\nSpatial pattern analysis of COVID-19. A total of nine articles were identified that emphasized on identifying the spatial transmission pattern analysis of COVID-1914,16,19,62–67. So far, only one study had used geospatial analysis to identify Spatio-temporal clusters and prediction modeling for COVID-19 transmission. They utilized the Poisson probability distribution model, Kernel density analysis, and space-time scanning analysis to identify high-risk Spatio-temporal clusters for transmission of COVID-19 in Brazil and detected active Spatio-temporal clusters comprising six municipalities in the south-central region of Brazil66. Only two studies so far studied how travel restrictions may have limited the epidemic trajectory. One of them developed a global model based on internationally reported cases and mobility data, to project the impact of travel limitations on the national and international spread of the epidemic and revealed that Wuhan travel ban only hindered the overall epidemic trajectory by 3 to 5 days in other cities of China but had a significant influence on the international scale dispersion22. Studies also estimated the probability of COVID-19 cases transportation before January 23 among 369 cities in China and reported that 130 cities in China have more than 50% chance of having a COVID-19 case imported from Wuhan67.\n\nIn a global study to identify geographic risks of COVID-19 transmission using online Rasch Modeling Algorithm, the authors reported that Iran, South Korea, Italy, Germany, Spain, China (Hubei), and France, are the top countries with higher outbreak potential respectively68. Other studies utilized accessibility analysis techniques to assess the spatial diffusion of COVID-19, using GIS-based origin, destinations, and frequencies mapping of public transportation from Wuhan. The study claimed that increase of COVID-19 cases have a direct relationship with the frequency of public transport from Wuhan69. Other studies utilized time-series analysis and plots to portray the spatial and temporal variation of COVID-19 cases in China and to elucidate the role of case importation in transmission in cities across China using spatial analysis70.\n\nOne other global study used GIS to visualize the worldwide distribution of air transport passengers from Wuhan and infected traveler’s ratio around the world19. Geo-referencing of confirmed cases also played an interesting role in the spatial pattern analysis studies. These studies measured and identified the regions that have a high risk of transmission at an early stage71. Similarly, a generalized linear regression model was used to analyze the spread and control of COVID-19 cases using case reports, and human movement data. This study utilized spatial distribution mapping of the travel movements from Wuhan to each province and modeled the spatial dispersal pattern of COVID-19 trajectories with or without Wuhan travel ban. They found that Wuhan shutdown delayed arrival of COVID-19 in other cities by around three days18.\n\nComputer-aided spatial and statistical analysis and modeling. A substantial number of studies were found to apply computer-aided spatial and statistical analysis and modeling techniques in analyzing distinct aspects of COVID-19. A total of 16 articles were identified that emphasized on computer-aided spatial and statistical analysis and modeling in COVID-1969–84. Most of these works were focused on analyzing the spatial distribution pattern of COVID-19 cases using the confirmed cases data72–78, or news reports of COVID-19 cases as proxy data79. Most of the studies were focused on examining the spatial distribution and relationship between COVID-19 cases, deaths, and locations. Adekunle et al. (2020) examined the same relationship. They found a positive statistically significant relationship among spatial clusters, confirmed cases and potential deaths72. Another similar study in Hubei, China, demonstrated that the high-low cluster had no high-value incidence cluster where local Moran's I indicated that Hubei was the only province with High-Low aggregation74. Moran's I was applied in analyzing the spatial and temporal distribution of cases in Hubei province by other studies as well77.\n\nMultiple studies examined the spatial and temporal distribution and modeled the trend of COVID-19 cases growth in ArcGIS and found that the highest risk place was those that had a high population inflow from Wuhan and Hubei province75,76. Spatial modeling using Poisson space-time scan statistics to produce cluster mapping attempts also led to the proposal of first rapid surveillance to monitor the spread of COVID-1973. Miller et al. (2020) demonstrated the worldwide spatial distribution of COVID-19 cases using the heat map technique in GIS The map reported that China, Italy, Iran, and Spain were the highest affected countries till March 17, 2020, which was also visible by the JHU and WHO reported data78. Spatial panel data model used by Guliyev (2020) showed that the rate of deaths had a significant positive effect where the recovery rate had a negative with the confirmed COVID-19 cases80. Irvine et al. (2020) used a SEIR model to estimate the transmission rate within Immigration and Customs Enforcement detention facilities in the GIS and the impacts on the I.C.U. capacity81.\n\nBai (2020) used two different models, including SEIRD model and Agent-Based Model (A.B.M.) to simulate the COVID-19 spread. They found that A.B.M. could be more effective and it also could be a useful tool to figure out new effective strategies82. In a similar work, Mollalo et al. (2020) compared five different models to develop a spatial model of COVID-19 incidence rate considering 35 variables using geospatial software’s. The results showed that MGWR could be a better model as it was able explain 68.1% of the total variation of COVID-19 incidences in the GIS83. In a follow-up paper, the same authors used ANN to model the incidence rate of COVID-19 and used Moran's I index to create incidence hotspots. Out of included 57 variables, 10 variables found statistically significant in explaining the result84. A similar global scale study revealed that age and population density have a statistically significant relationship with the spatial distribution pattern of COVID-19 cases85.\n\nUsing Maxent based Ecological Niche Model, Ren et al. (2020) developed a potential risk zone map of China where population, public transportation demands, medical resources demands were used as explanatory variables. They suggested using this as an early forecasting model to predict the risk zone in China's other megacities86. Kanga et al. (2020), on the other hand, provided a useful recommendation to local authorities in India by using proximity-based hot spot analysis to map the risk zones with relevant preventative measures to mitigate the COVID-19 crisis87. Several other works were focused on suitability mapping with a focus to find a suitable location for the quarantine zone in Surat, India, using ArcGIS. The study revealed that the suitability analysis could help to control the spread as a prevention measure.\n\n\nData mining and COVID-19\n\nWe grouped the application of big data, social media data mining, and contact tracing through geospatial technologies together. Although very few studies attempted to incorporate those techniques in COVID-19 research, a total of eight articles were identified that emphasized on data mining, big data, and social media data use in COVID-1962,85–91. Data mining using unsupervised machine learning models were utilized in a study to analyze the twitter data and it reported tweets related to symptoms of users is associated with COVID-19 testing accessibility66. Studies also used nighttime light (NTL) data and AQI data to analyze the spatial and temporal pattern of COVID-19 and how that impacted human activities. The observation demonstrated that the NTL brightness and AQI value were much lower during the quarantine period in Mainland China88. Similarly, radar data was used to detect traffic patterns where the findings reported that the number of heavy vehicles movement in the region changed significantly after the COVID outbreak89.\n\nSeveral studies used mobile sensor data or geodata for contact tracing as a surveillance strategy to monitor COVID-1990–92. Wang et al. (2020) developed a Geo-AI based mini program within an instant messaging app (WeChat) to trace close contacts of all confirmed patients. The results showed that the program could analyze real-time data to trace the contacts, and those data could be used with other datasets to find out more useful information to reduce COVID-19 spread. Similarly, other studies also attempted to develop a smart contact tracing app using big data analytics90 or using a mobile sensor-based contact tracing system to minimize the spread of COVID-1991. Using social media posts, Huang et al. (2020) examined the attributes of both suspected and confirmed COVID-19 cases who contacted with the symptoms. They used SPSS and ArcGIS for descriptive statistical analysis and spatial analysis, where they found that most of the patients seeking help were above 65 years old from Wuhan93.\n\n\nDiscussion\n\nTo the best of our knowledge, this is one of the first systematic reviews of the application of GIS and other geospatial technologies in COVID-19 related research. Our work does not only provide an overview of how GIS was used so far but also provides pointers on how GIS could be more efficiently used in COVID-19-related works and other public health issues in the coming days. The application of GIS technologies and spatial analysis has substantially influenced the understanding of COVID-19, not only for the scientific community but also for the policymakers, and for the public in building a long term response to the ongoing pandemic94. Initially, spatial analysis techniques were used as part of predictive modeling to predicts the growth of COVID-19 cases63 and to model the spatio-temporal variation of confirmed incidences74. With the increasing availability of COVID-19 data, a significant number of studies started to analyze the spatial transmission pattern and spread of the virus from Wuhan to other cities in China and the rest of the world17,18,20,22,26,67,71,89,95. Most of these early applications of GIS and spatial analysis were more focused on visualizing the COVID-19 confirmed cases as well as the distribution of cases among administrative units and countries. However, as time goes on and more data became available, more complex GIS tools come into play. Studies not only used GIS for analyzing different environmental aspects; they also used different earth observation data acquired by the European Space Agency (ESA) and NASA23,24,28,34.\n\nDuring the early days, one of the biggest discussions among the researchers was regarding the ability of meteorological factors to limit the spread of coronavirus. With the declaration of the pandemic and subsequent lockdown globally in early March, several studies used GIS and remotely sensed images to analyze the impacts of this lockdown on the environment, air quality, and other particulate matters18,23,26. Several studies evaluated that relationship with the help of spatial analytical tools, and most of these studies could not reach a valid conclusion where they could claim temperature or other climatic factors do limit the spread of the virus23,24,27,30. Later application of GIS does not restrict itself in just visualizing. Instead, it was more used to spatial autocorrelation and clustering analysis, hotspot analysis, and suitability analysis to see whether any association exists among social and economic groups, any specific location or social group, and COVID-19 infection rate55,56,59. With the increasing confirmed cases around the world, different social-science studies analyzed the association among COVID-19 infection rate and social vulnerability, racial inequality, risk perception, resiliency, and settlement quality issues44,45,47. These studies also utilized GIS to identify any spatial patterns and autocorrelation with COVID-19.\n\nWe reviewed how GIS and spatial analysis techniques were used in the past COVID-19 related studies. We found that most of the included studies used GIS for visualizing the spatial distribution and pattern of COVID-19 spread, cluster analysis to identify the accumulation of cases, hot spot analysis to find out any outbreak, proximity analysis to evaluate the accessibility to the primary health care facilities. However, with time, studies focused on different models to predict or simulate various aspects of COVID-19 using geospatial techniques that were published as well. On that point of interest, GIS-based Maxent model, spatial data panel model, SEIR model, Agent-Based Model, GWR, MGWR, ANN were used in different studies80–84,86. However, we did not find any studies that used spatially explicit modeling to identify and predict the location of any potential outbreak in the future. One of the few positive aspects of COVID-19-related studies is the publicly available data, and the same was noticed in the review as well. We found that more than half of the studies used data from some form of government database followed by WHO database (n=12), different websites (n=12), JHU dashboard and Worldometers (n=9), Social media data (n=8), satellite images (n=7), primary survey (n=3) and mobile phone data (n=3).\n\nThough COVID-19 related data is mostly publicly available, some studies reported data unavailability issues, especially in developing or less developed countries32,49,84. The major challenge of global or regional studies is the possibility of an under-reported number of confirmed cases, especially in low-income regions, because of the low detection coverage of COVID-19, which may skew the result. Most of the global or regional studies cannot incorporate the controlling measures imposed by different governments, which has significant impacts on the spread and infection incidences of COVID-19 cases. No consideration of government control measures and low testing issues is also a substantial limitation for modeling and prediction focused research which creates a biased result. Therefore, future studies should emphasize considering government control measures and policies in their modeling. Although contract tracing and data mining research has been proven to be useful in analyzing and forecasting the spatial pattern of COVID dispersion, no trace of any studies were found outside of China and the USA. That might be due to data unavailability and technological issues. Contract tracing in China was possible due to its government-backed app that gathers a user's information, including name, ID number, and health information and movement data. Two studies were conducted in China and Taiwan for smart contract tracing using mobile sensor data90,92. Low or middle-income countries can adopt a GIS-based volunteered surveillance approach where peoples will share their information voluntarily to tackle the pandemic. So far, GIS has not been used much to track the transmission pattern and to predict the transmission. That is something that can be done at a global level to leverage GIS to predict not only the confirmed case numbers but also specific locations where the outbreak would happen with higher statistical precision96.\n\nThe findings of this review have profound implications for contemporary and future multidisciplinary scientific research, policymaking, and practice. The diverse use of GIS technologies in different overarching thematic areas of scientific research highlights the potential of incorporating methodological perspectives for solving complex research questions. This evolution is consistent with the emerging perspective that “one size does not fit all” and each unique scenario may require conceptual and empirical inputs from different disciplines for achieving a higher precision on research outcomes. Nonetheless, an increasing trend of integrating GIS technologies in studies that emphasized on multiple research objectives show how such technologies are being a part of the entire work rather than the only approach used in those research efforts. Thus, the use of GIS may improve other methodological measures and increase the scope of scientific exploration on a topic of interest.\n\nThe existing evidence highlights the use of GIS and other geospatial techniques for addressing research question; however, little evidence exists on how geospatial can be used for delivering digital interventions for individuals or target populations. Perhaps such technological innovations would take much time to appear, but precision sciences and their applications on personalizing user level platforms may bring such technologies more closer to everyday practice. Moreover, a wide range of data sources used in different studies included in this review provide meaningful insights on how data from multiple can be harmonized and utilized in addressing population-based problems. Furthermore, integrating GIS in COVID-19 related research may enable real-time decision-making for preventing public health crises and deploying resources whenever required. A major lesson from existing studies is to developing local and global disaster preparedness plans that may enable policymakers and practitioners to leverage GIS-based advanced data analytics for mitigating large scale public health emergencies. More implementation research is needed to assess the scope of such multipronged yet coordinated response systems that may emerge in the post-pandemic world. Such initiatives may require strengthening technological capacities in low and middle-income countries that share a major proportion of global health problems, yet have limited resources to address the same97,98.\n\nDespite notable strengths, this systematic review has several limitations that should be acknowledged and addressed in future research. First, the selection of databases and keywords could have excluded some studies that were indexed in other databases or used non-specified keywords, which were beyond the scope of this review. Second, we focused on peer-reviewed publications and did not cover preprints that did not undergo peer-review; therefore, those studies are also excluded from this review, which may provide further insights on the evidence landscape. Third, the existing literature shows a high heterogeneity in the methods, data inputs, and research outcomes leading to a narrative synthesis. Fourth, we did not assess the quality of the studies and the risk of bias within and between the studies. We recommend that future evidence syntheses on specific GIS-related topics should assess the risk of bias among the scientific literature in those topics. Prospective evidence-based reviews may also consider the quantitative synthesis of homogenous studies on specific themes. This systematic review provides an inclusive and extensive synthesis of multidisciplinary research using GIS during COVID-19, which may inform future primary studies and advanced syntheses addressing the current limitations and improving the knowledge base in this domain.\n\n\nConclusion\n\nThis systematic review evaluated the current literature on the use of GIS and geospatial analyses in the context of COVID-19 pandemic and explored the scope of integrating such techniques in the current research efforts as well as future research and practice. In the era of digital revolution, a growing need for exchanging technological advancements across scientific disciplines is widely acknowledged. The use of GIS and related technologies in COVID-19 pandemic examplifies such integrations and provide scholarly perspectives on how complex societal and global issues can be understood using the existing tools. Moreover, such applications necessitate revisiting the current strengths and weaknesses of curating evidence across contexts. It is essential to strengthening institutional capacities to leverage GIS-related technologies in multipronged research and development that empower research communities to work together in this pandemic. Last but not least, future technological innovations should be grounded on the lessons learned during this pandemic to make such technologies readily available for facilitating robust research and decision-making that may improve population-level outcomes globally.\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: Application of geospatial techniques in COVID-19-related studies. https://doi.org/10.6084/m9.figshare.13229147.v216.\n\nThis project contains a summary of the articles idenitifed in this study. This includes a detailed breakdown of what methods used in the reviewed article, along with the data type, spatial analysis tool/techniques and the findings from the analysis.\n\nOpen Science Framework: PRISMA checklist for ‘Applications of GIS and geospatial analyses in COVID-19 research: A systematic review’. https://doi.org/10.17605/OSF.IO/ZGMP899.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0); the compelted PRISMA checklist is available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWorld Health Organization (WHO): Coronavirus disease 2019 (‎COVID-19)‎: situation report, 201. 2020a. Reference Source\n\nMcNeil MG Jr: Coronavirus Has Become a Pandemic, W.H.O. Says. in The New York Times. ed. New York: The New York Times Company, 2020. Reference Source\n\nWorld Health Organization (WHO): Coronavirus disease 2019 (‎COVID-19)‎: situation report, 250. 2020b.\n\nDong E, Du H, Gardner L: An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect Dis. 2020; 20(5): 533–534. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFranch-Pardo I, Napoletano BM, Rosete-Verges F, et al.: Spatial analysis and GIS in the study of COVID-19. A review. Sci Total Environ 2020; 739: 140033. PubMed Abstract \| Publisher Full Text\n\nBoulos MNK, Geraghty EM: Geographical tracking and mapping of coronavirus disease COVID-19/severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic and associated events around the world: how 21st century GIS technologies are supporting the global fight against outbreaks and epidemics. (in eng). Int J Health Geogr. 2020; 19(1): 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nESRI: History of GIS. (accessed July 29, 2020, 2020). Reference Source\n\nLyseen AK, Nøhr C, Sørensen EM, et al.: A Review and Framework for Categorizing Current Research and Development in Health Related Geographical Information Systems (GIS) Studies. (in eng). Yearb Med Inform. 2014; 9(1): 110–124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopkins J: CSSE Coronavirus COVID-19 Global Cases (dashboard). (accessed August 24, 2020). Reference Source\n\nHopkins J: CSSE GitHub–CSSEGISandData/COVID-19: Novel Coronavirus (COVID-19) Cases (data repository). (accessed August 9, 2020). Reference Source\n\nTexas Department of State Health Services: Texas Case Counts- COVID-19. accessed. Reference Source\n\nFranch-Pardo I, Napoletano BM, Rosete-Verges F, et al.: Spatial analysis and GIS in the study of COVID-19. A review. Sci Total Environ. 2020; 739: 140033. PubMed Abstract \| Publisher Full Text\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. J Clin Epidemiol. 2009; 62(10): e1–e34. PubMed Abstract \| Publisher Full Text\n\nPopay J, et al.: Guidance on the conduct of narrative synthesis in systematic reviews. A product from the ESRC methods programme Version. 2006; 1: b92. Publisher Full Text\n\nAhasan R, Alam MS, Chakraborty T, et al.: GIS in COVID-19 research. 2020. Publisher Full Text\n\nAhasan R, Alam MS, Chakraborty T, et al.: Application of geospatial techniques in COVID-19 related studies. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.13229147.v2\n\nKraemer MU, Yang CH, Gutierrez B, et al.: The effect of human mobility and control measures on the COVID-19 epidemic in China. Science. 2020; 368(6490): 493–497. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTian H, Liu Y, Li Y, et al.: An investigation of transmission control measures during the first 50 days of the COVID-19 epidemic in China. Science. 2020; 368(6491): 638–642. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChristidis P, Christodoulou A: The Predictive Capacity of Air Travel Patterns during the Global Spread of the COVID-19 Pandemic: Risk, Uncertainty and Randomness. Int J Environ Res Public Health. 2020; 17(10): 3356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoulos MNK, Geraghty EM: Geographical tracking and mapping of coronavirus disease COVID-19/severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic and associated events around the world: how 21st century GIS technologies are supporting the global fight against outbreaks and epidemics. Int J Health Geogr. 2020; 19(1): 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPequeno P, Mendel B, Rosa C, et al.: Air transportation, population density and temperature predict the spread of COVID-19 in Brazil. PeerJ. 2020; 8: e9322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChinazzi M, Davis JT, Ajelli M, et al.: The effect of travel restrictions on the spread of the 2019 novel coronavirus (COVID-19) outbreak. Science. 2020; 368(6489): 395–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLal P, Kumar A, Kumar S, et al.: The dark cloud with a silver lining: Assessing the impact of the SARS COVID-19 pandemic on the global environment. Sci Total Environ. 2020; 732: 139297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuhammad S, Long X, Salman M: COVID-19 pandemic and environmental pollution: a blessing in disguise? Sci Total Environ. 2020; 728: 138820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIqbal MM, Abid I, Hussain S, et al.: The effects of regional climatic condition on the spread of COVID-19 at global scale. Sci Total Environ. 2020; 739: 140101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaker RE, Yang W, Vecchi GA, et al.: Susceptible supply limits the role of climate in the early SARS-CoV-2 pandemic. Science. 2020; 369(6501): 315–319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu Y, Jing W, Liu J, et al.: Effects of temperature and humidity on the daily new cases and new deaths of COVID-19 in 166 countries. Sci Total Environ. 2020; 729: 139051. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanniah KD, Zaman NAFK, Kaskaoutis DG, et al.: COVID-19's impact on the atmospheric environment in the Southeast Asia region. Sci Total Environ. 2020; 736: 139658. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDepellegrin D, Bastianini M, Fadini A, et al.: The effects of COVID-19 induced lockdown measures on maritime settings of a coastal region. Sci Total Environ. 2020; 740: 140123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBriz-Redón Á, Serrano-Aroca Á: A spatio-temporal analysis for exploring the effect of temperature on COVID-19 early evolution in Spain. Sci Total Environ. 2020; 728: 138811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRunkle JD, Sugg MM, Leeper RD, et al.: Short-term effects of specific humidity and temperature on COVID-19 morbidity in select US cities. Sci Total Environ. 2020; 740: 140093. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu H, Yan C, Fu Q, et al.: Possible environmental effects on the spread of COVID-19 in China. Sci Total Environ. 2020; 731: 139211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Z, Xue T, Jin X: Effects of meteorological conditions and air pollution on COVID-19 transmission: Evidence from 219 Chinese cities. Sci Total Environ. 2020; 741: 140244. PubMed Abstract \| Publisher Full Text\n\nFan C, et al.: The Impact of the Control Measures during the COVID-19 Outbreak on Air Pollution in China. Remote Sensing. 2020; 12(10): 1613.\n\nAhmadi M, Sharifi A, Dorosti S, et al.: Investigation of effective climatology parameters on COVID-19 outbreak in Iran. Sci Total Environ. 2020; 729: 138705. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta S, Raghuwanshi GS, Chanda A: Effect of weather on COVID-19 spread in the US: A prediction model for India in 2020. Sci Total Environ. 2020; 728: 138860. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQi H, Xiao S, Shi R, et al.: COVID-19 transmission in Mainland China is associated with temperature and humidity: A time-series analysis. Sci Total Environ. 2020; 728: 138778. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArab-Mazar Z, Sah R, Rabaan AA, et al.: Mapping the incidence of the COVID-19 hotspot in Iran - Implications for Travellers. Travel Med Infect Dis. 2020; 34: 101630. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBashir MF, Ma B, Bilal, et al.: Correlation between climate indicators and COVID-19 pandemic in New York, USA. Sci Total Environ. 2020; 728: 138835. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBao R, Zhang A: Does lockdown reduce air pollution? Evidence from 44 cities in northern China. Sci Total Environ. 2020; 731: 139052. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahato S, Pal S, Ghosh KG: Effect of lockdown amid COVID-19 pandemic on air quality of the megacity Delhi, India. Sci Total Environ. 2020; 730: 139086. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShahzad F, Shahzad U, Fareed Z, et al.: Asymmetric nexus between temperature and COVID-19 in the top ten affected provinces of China: A current application of quantile-on-quantile approach. Sci Total Environ. 2020; 736: 139115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKerimray A, Baimatova N, Ibragimova OP, et al.: Assessing air quality changes in large cities during COVID-19 lockdowns: The impacts of traffic-free urban conditions in Almaty, Kazakhstan. Sci Total Environ. 2020; 730: 139179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu J, Zhou J, Yao J, et al.: Impact of meteorological factors on the COVID-19 transmission: A multi-city study in China. Sci Total Environ. 2020; 726: 138513. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYao Y, Pan J, Wang W, et al.: Association of particulate matter pollution and case fatality rate of COVID-19 in 49 Chinese cities. Sci Total Environ. 2020; 741: 140396. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaez A, Lopez FA, Menezes T, et al.: A Spatio-Temporal Analysis of the Environmental Correlates of COVID-19 Incidence in Spain. Geogr Anal. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaraye IM, Horney JA: The Impact of Social Vulnerability on COVID-19 in the U.S.: An Analysis of Spatially Varying Relationships. Am J Prev Med. 2020; 59(3): 317–325. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim SJ, Bostwick W: Social Vulnerability and Racial Inequality in COVID-19 Deaths in Chicago. Health Educ Behav. 2020; 47(4): 509–513. PubMed Abstract \| Publisher Full Text\n\nRamírez IJ, Lee J: COVID-19 Emergence and Social and Health Determinants in Colorado: A Rapid Spatial Analysis. Int J Environ Res Public Health. 2020; 17(11): 3856. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang CH, Schwartz GG: Spatial Disparities in Coronavirus Incidence and Mortality in the United States: An Ecological Analysis as of May 2020. J Rural Health. 2020; 36(3): 433–445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGibson L, Rush D: Novel Coronavirus in Cape Town Informal Settlements: Feasibility of Using Informal Dwelling Outlines to Identify High Risk Areas for COVID-19 Transmission From A Social Distancing Perspective. JMIR Public Health Surveill. 2020; 6(2): e18844. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLakhani A: Introducing the Percent, Number, Availability, and Capacity [PNAC] Spatial Approach to Identify Priority Rural Areas Requiring Targeted Health Support in Light of COVID-19: A Commentary and Application. J Rural Health. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLakhani A: Which Melbourne Metropolitan Areas Are Vulnerable to COVID-19 Based on Age, Disability, and Access to Health Services? Using Spatial Analysis to Identify Service Gaps and Inform Delivery. J Pain Symptom Manage. 2020: 60(1): e41–e44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDryhurst S, et al.: Risk perceptions of COVID-19 around the world. Journal of Risk Research. 2020; 1–13. Publisher Full Text\n\nSarkar SK: COVID-19 Susceptibility Mapping Using Multicriteria Evaluation. Disaster Med Public Health Prep. 2020; 1–17. PubMed Abstract \| Publisher Full Text\n\nCavalcante JR, Lopes de Abreu AdJ: COVID-19 in the city of Rio de Janeiro: spatial analysis of first confirmed cases and deaths. Epidemiol Serv Saude. 2020; 29(3): e2020204. PubMed Abstract \| Publisher Full Text\n\nRubino I, Coscia C, Curto R: Identifying Spatial Relationships between Built Heritage Resources and Short-Term Rentals before the Covid-19 Pandemic: Exploratory Perspectives on Sustainability Issues. Sustainability. 2020; 12(11): 4533. Publisher Full Text\n\nRequia WJ, Kondo EK, Adams MD, et al.: Risk of the Brazilian health care system over 5572 municipalities to exceed health care capacity due to the 2019 novel coronavirus (COVID-19). Sci Total Environ. 2020; 730: 139144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJovanović A, et al.: Assessing resilience of healthcare infrastructure exposed to COVID-19: emerging risks, resilience indicators, interdependencies and international standards. Environment Systems & Decisions. 2020; 1. Publisher Full Text\n\nJella TK, Acuña AJ, Samuel LT, et al.: Geospatial mapping of orthopaedic surgeons age 60 and over and confirmed cases of COVID-19. J Bone Joint Surg Am. 2020; 102(12): 1022–1028. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuthberg JS, et al.: Geospatial analysis of COVID-19 and otolaryngologists above age 60. Am J Otolaryngol. 2020; 14(4): 102514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuupiel D, et al.: Geographical accessibility to glucose-6-phosphate dioxygenase deficiency point-of-care testing for antenatal care in Ghana. Diagnostics (Basel). 2020; 10(4): 229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhmadi A, Fadai Y, Shirani M, et al.: Modeling and forecasting trend of COVID-19 epidemic in Iran until May 13, 2020. Med J Islam Repub Iran. 2020; 34(1): 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFan J, Liu X, Pan W, et al.: Epidemiology of Coronavirus Disease in Gansu Province, China, 2020. Emerg Infect Dis. 2020; 26: 1257–1265. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHart OE, Halden RU: Computational analysis of SARS-CoV-2/COVID-19 surveillance by wastewater-based epidemiology locally and globally: Feasibility, economy, opportunities and challenges. Sci Total Environ. 2020; 730: 138875. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrade LA, et al.: Surveillance of the first cases of COVID-19 in Sergipe using a prospective spatiotemporal analysis: the spatial dispersion and its public health implications. Rev Soc Bras Med Trop. 2020; 53: e20200287. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDu Z, et al.: Risk for transportation of coronavirus disease from Wuhan to other cities in China. Emerg Infect Dis. 2020; 26(5): 1049–1052. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJen T-H, Chien T-W, Yeh Y-T, et al.: Geographic risk assessment of COVID-19 transmission using recent data: An observational study. Medicine (Baltimore). 2020; 99(24): e20774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng R, Xu Y, Wang W, et al.: Spatial transmission of COVID-19 via public and private transportation in China. Travel Med Infect Dis. 2020; 34: 101626. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang D, Choi H, Kim J-H, et al.: Spatial epidemic dynamics of the COVID-19 outbreak in China. Int J Infect Dis. 2020; 96–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJia JS, Lu X, Yuan Y, et al.: Population flow drives spatio-temporal distribution of COVID-19 in China. Nature. 2020; 582(7812): 389–94. PubMed Abstract \| Publisher Full Text\n\nAdekunle IA, Onanuga A, Wahab O, et al.: Modelling spatial variations of coronavirus disease (COVID-19) in Africa. Sci Total Environ. 2020; 729: 138998. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDesjardins M, Hohl A, Delmelle E: Rapid surveillance of COVID-19 in the United States using a prospective space-time scan statistic: Detecting and evaluating emerging clusters. Appl Geogr. 2020; 118: 102202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Li H, Ding Z, et al.: Spatial statistical analysis of Coronavirus Disease 2019 (Covid-19) in China. Geospat Health. 2020; 15(1). PubMed Abstract \| Publisher Full Text\n\nYe L, Hu L: Spatiotemporal distribution and trend of COVID-19 in the Yangtze River Delta region of the People’s Republic of China. Geospatial Health. 2020; 15(1). PubMed Abstract \| Publisher Full Text\n\nTang W, Liao H, Wang Z, et al.: The Changing Patterns of Coronavirus Disease 2019 (COVID-19) in China: A Tempogeographic Analysis of the Severe Acute Respiratory Syndrome Coronavirus 2 Epidemic. Clin Infect Dis. 2020; 71(15): 818 –824. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang W, Deng M, Li C, et al.: Spatio-Temporal Patterns of the 2019-nCoV Epidemic at the County Level in Hubei Province, China. Int J Environ Res Public Health. 2020; 17(7): 2563. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller LE, Bhattacharyya R, Miller AL: Spatial Analysis of Global Variability in Covid-19 Burden. Risk Manag Healthc Policy. 2020; 13: 519–522. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThakar V: Unfolding Events in Space and Time: Geospatial Insights into COVID-19 Diffusion in Washington State during the Initial Stage of the Outbreak. ISPRS Int J Geoinf. 2020; 9(6): 382. Publisher Full Text\n\nGuliyev H: Determining the spatial effects of COVID-19 using the spatial panel data model. Spat Stat. 2020; 100443. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIrvine M, Coombs D, Skarha J, et al.: Modeling COVID-19 and Its Impacts on US Immigration and Customs Enforcement (ICE) Detention Facilities, 2020. J Urban Health. 2020; 97(4): 439–447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBai S: Simulations of COVID-19 spread by spatial agent-based model and ordinary differential equations. International Journal of Simulation and Process Modelling. 2020; 15(3): 268–277. Publisher Full Text\n\nMollalo A, Vahedi B, Rivera KM: GIS-based spatial modeling of COVID-19 incidence rate in the continental United States. Sci Total Environ. 2020; 728: 138884. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMollalo A, Rivera KM, Vahedi B: Artificial Neural Network Modeling of Novel Coronavirus (COVID-19) Incidence Rates across the Continental United States. Int J Environ Res Public Health. 2020; 17(12): 4204. 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Publisher Full Text\n\nTanveer H, Balz T, Cigna F, et al.: Monitoring 2011–2020 Traffic Patterns in Wuhan (China) with COSMO-SkyMed SAR, Amidst the 7th CISM Military World Games and COVID-19 Outbreak. Remote Sensing. 2020; 12(10): 1636. Publisher Full Text\n\nChen CM, Jyan HW, Chien SC, et al.: Containing COVID-19 Among 627,386 Persons in Contact With the Diamond Princess Cruise Ship Passengers Who Disembarked in Taiwan: Big Data Analytics. J Med Internet Res. 2020; 22(5): e19540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEkong I, Chukwu E, Chukwu M: COVID-19 Mobile Positioning Data Contact Tracing and Patient Privacy Regulations: Exploratory Search of Global Response Strategies and the Use of Digital Tools in Nigeria. JMIR Mhealth Uhealth. 2020; 8(4): e19139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang S, Ding S, Xiong L: A New System for Surveillance and Digital Contact Tracing for COVID-19: Spatiotemporal Reporting Over Network and GPS. 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PubMed Abstract \| Publisher Full Text\n\nHossain MM, Tasnim T, Sharma R, et al.: Digital interventions for people living with non-communicable diseases in India: A systematic review of intervention studies and recommendations for future research and development. Digit Health. 2019; 5: 2055207619896153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhasan R, Alam S, Chakraborty T, et al.: GIS in COVID-19 research. 2020. http://www.doi.org/10.17605/OSF.IO/ZGMP8" }	[ { "id": "95380", "date": "25 Oct 2021", "name": "Hamid Mukhtar", "expertise": [ "Reviewer Expertise computer science", "information visualization", "data analytics", "machine learning", "deep learning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article reviews existing approaches in the use of geographical information systems (GIS) for COVID-19 containment, contact tracing, surveillance and hotspots identification. Further, it suggests how GIS can be used by the scientific community and policy-makers for informed decision-making.\nThe article uses the PRISMA approach for systematic analysis of the application of GIS in COVID-19. The approach is explained clearly. The outcome of the study with various dimensions has been discussed. The limitations have also been discussed.\nThe MEDLINE and Web of Science repositories were used as data sources. My concern is that there are GIS specific journals and conferences organized by organizations like IEEE and ACM; have authors tried to explore those venues for better results? Why not? Apparently, GIS in COVID-19 should be searchable in computing and engineering databases rather than medical databases.\nOn the positive side, the authors have created thematic groups from the review, which is useful for readers.\nI suggest that the authors add a summary table that can provide the considered dimensions, attributes, considerations, etc. Figure 2 may be reconsidered without the world map.\nThe quality of figure 1 is not very good, should be improved.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "97367", "date": "16 Dec 2021", "name": "Orhun Aydin", "expertise": [ "Reviewer Expertise Spatial statistics", "GIS", "space-time statistics", "Earth science", "human-environment interaction", "geostatistics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe sentence “Although GIS has substantial potential in planning to slow down the spread, surveillance, contact tracing, and identify the trends and hotspots of breakdowns, it was not employed as much as it could have been.” Can be expanded. This sentence can also include another vital role that GIS played, putting data on a map to inform the public. WebGIS was particularly crucial during the pandemic for keeping the public informed of the spread of COVID-19.\nIn the introduction the sentence “Although the first reported case was in China, and it was the epicenter of the pandemic, the virus has mutated and changed transmission pattern several times since then.” needs a reference.\nThe sentence \"Besides RStudio, QGIS (n=4) is another open source software platform that was highly employed by the researchers.\" RStudio is a software for programming whereas QGIS is a software platform for GIS\". I think QGIS and ArcGIS would fare better if they were in the same sentence.\nParagraph starting with \"ESRI ArcGIS is the most used platform\" has to mention Python. Most labs forecasting the spread of the virus IHME, CHIME to name a few, provided Python libraries and they were extensively used by decision-makers.\nSIGSPATIAL had two conferences on COVID. Below are references from the main paper and one of our contributions. Note that in the statement \"SEIR model, Agent-Based Model, GWR, MGWR, ANN were used in different studies\" you will see in this workshop that there were studies that used GIS and SEIR, SIR-type epidemiological models.\nWhen data challenges are discussed an important point to inform the reader on would be confidentiality.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "7734", "date": "24 Jan 2022", "name": "Rakibul Ahasan", "role": "Author Response", "response": "We thank you for your comments on our manuscript. We believe addressing the comments helped to improve the quality of our work. Please find the point-by-point responses and revision details below- The sentence “Although GIS has substantial potential in planning to slow down the spread, surveillance, contact tracing, and identify the trends and hotspots of breakdowns, it was not employed as much as it could have been.” Can be expanded. This sentence can also include another vital role that GIS played, putting data on a map to inform the public. WebGIS was particularly crucial during the pandemic for keeping the public informed of the spread of COVID-19. GIS maps, especially web maps and WebGIS-based dashboards, played an important role in informing the public regarding the spread of the virus, especially during the early days of the pandemic. Therefore, in addition to our previous statement, we have included Geospatial techniques, especially WebGIS, which have even been widely used in putting the data into a map and were critical to informing the public regarding the spread of the virus especially during the early days of the pandemic. In the introduction the sentence “Although the first reported case was in China, and it was the epicenter of the pandemic, the virus has mutated and changed transmission pattern several times since then.” needs a reference. We included references to support our statement regarding the first reported case. We used one of the most evidence-based, peer-reviewed works we could find in PubMed (Alam, 2020). For the virus mutation and transmission pattern, we used a reference that discussed the chronological mutation of the virus- from the original variant to the most recent Omicron variant (Kannan et al., 2022). Allam Z. (2020). The First 50 days of COVID-19: A Detailed Chronological Timeline and Extensive Review of Literature Documenting the Pandemic. Surveying the Covid-19 Pandemic and its Implications, 1–7. https://doi.org/10.1016/B978-0-12-824313-8.00001-2 Kannan, S. R., Spratt, A. N., Sharma, K., Chand, H. S., Byrareddy, S. N., & Singh, K. (2022). Omicron SARS-CoV-2 variant: Unique features and their impact on pre-existing antibodies. Journal of autoimmunity, 126, 102779. https://doi.org/10.1016/j.jaut.2021.102779 The sentence \"Besides RStudio, QGIS (n=4) is another open-source software platform that was highly employed by the researchers.\" RStudio is a software for programming whereas QGIS is a software platform for GIS\". I think QGIS and ArcGIS would fare better if they were in the same sentence. We initially grouped QGIS with RStudio as both are free and open-source software. However, we agree based on the functionality, QGIS better fits with ESRI ArcGIS. Therefore, we changed the sentences accordingly. Paragraph starting with \"ESRI ArcGIS is the most used platform\" has to mention Python. Most labs forecasting the spread of the virus IHME, CHIME to name a few, provided Python libraries and they were extensively used by decision-makers. SIGSPATIAL had two conferences on COVID. Below are references from the main paper and one of our contributions. Note that in the statement \"SEIR model, Agent-Based Model, GWR, MGWR, ANN were used in different studies\" you will see in this workshop that there were studies that used GIS and SEIR, SIR-type epidemiological models. As the latest date of our review was June 28, 2020, papers published after that date were out of our review's scope. However, we strongly agree it is important to discuss these epidemiological models that were used to forecast the spread of the virus within a geographic context. Therefore, we revised the discussion section to include the use of epidemiological models. At the same time, we believe future systematic reviews would include these works that were published later, which was beyond the scope of our review. When data challenges are discussed an important point to inform the reader on would be confidentiality. This is an important point. Data confidentiality has been widely discussed not only in public health research but also in other domains as well. Even geospatial techniques using satellite images and remote sensing has been facing backlashes over data confidentiality issues. We call for robust data management and sharing practices ensuring confidentiality and ethical use of data. We express gratitude to the reviewer for their time and valuable comments and hope this updated, revised version of our work is more comprehensive and satisfactory." } ] } ]	1	https://f1000research.com/articles/9-1379
https://f1000research.com/articles/11-116/v1	28 Jan 22	{ "type": "Research Article", "title": "Towards an interprofessional competency-based model in South Africa: A Delphi study", "authors": [ "Gérard Charl Filies", "José Frantz", "José Frantz" ], "abstract": "Background: The overall objective of any health professional curriculum is to ensure that the knowledge, skills and attitudes of the students are influenced by the curriculum and to instil these attributes into the students, to help them become capable, compassionate and inquisitive health professionals. Therefore, there is the need for medical educators to align their learning objectives with the core competencies needed to achieve this. Currently, in interprofessional education, it is not always clear which activities may be used to facilitate the development of interprofessional core competencies. However, if health professional students are exposed to the interprofessional core competencies effectively, it may result in health care professionals who have an improved understanding of interprofessional practices, thus improving these practices within their specific professions. The objective of this study was to identify teaching strategies and activities that aim to develop interprofessional competencies in undergraduate health care students at the University of the Western Cape, South Africa. Methods: In this study the Delphi method was used to reach a consensus on the most appropriate activities and assessment methods to use in an interprofessional curriculum that would assist in instilling interprofessional core competencies in undergraduate health care students. Results: 19 experts out of 69 invited participants took part in this Delphi study. The activities highlighted were case studies, joint clinical placements, simulations, role plays and workshops/discussions. The assessment forms highlighted by participants were portfolios, reflection and rubrics. Conclusion: It was evident from this study that such activities can be used to instil more than one core competency in undergraduate health care students.", "keywords": [ "interprofessional education", "interprofessional core competencies", "graduate attributes", "curriculum transformation", "curriculum mapping" ], "content": "Introduction\n\nAny health professional university curriculum has an overall objective to ensure that the knowledge, skills and attitudes of the students are influenced by the curriculum and that attributes which would help students to be compassionate and inquisitive health professionals are instilled in them. McKean et al., highlights the need for medical educators to align their learning objectives with the core competencies of the role in question, if they strive to ensure that the students achieve a degree of competency.1 The limited studies in interprofessional education do highlight activities that may be used to facilitate the development of interprofessional core competencies, but no such studies have been conducted in South Africa.2,3 However, it is our understanding that, if health professional university students are exposed to the interprofessional core competencies effectively, it may result in health care professionals who have an improved understanding of interprofessional practices, thus improving practices within their specific professions. The Interprofessional Education Collaborative Expert Panel defines interprofessional core competencies as an integration of knowledge, skills, and values/attitudes that defines teamwork across the health professions and with patients, their families and communities they live in, with the intention of improving health outcomes.4 Interprofessional practice occurs when those health professionals from different disciplines work together with patients, families, carers, communities and each other to render comprehensive health care.5 For example, a person who recently had a stroke would be seen by a team of health professionals in one consultation for assessment and then planning a treatment programme with the client and their family and/or caregiver, instead of seeing each health professional separately.\n\nInterprofessional competencies are being used gradually more and more by many professions to comprehensively describe ideas such as interprofessional collaboration.6 Interprofessional collaboration takes place when more than two professionals work together to achieve common aims to solve a variety of complex challenges.7 For example, the Royal College of Physicians and Surgeons of Canada’s CanMEDS competency framework has been embraced by professions such as nurses, chiropractors, paramedics, physician assistants, family physicians and veterinarians at a global level.8\n\nIn this study, a systematic review was conducted to determine if any research studies have been done in an South African context, which explored how learning and teaching activities were used to develop core competencies among students.9–13 This study found that no studies in South Africa had been conducted, to the best of the authors’ knowledge. Internationally, only five studies were found that incorporate interprofessional core competency development into their programmes.14 Of these five studies, the higher education institutions did not include all competencies in their learning and teaching activities and there was no evidence of the impact of their programmes with regard to improving health outcomes for clients, patients, families or communities. This study therefore suggests that a transformative curriculum is required to reflect interprofessional core competency development in health professions’ training over the continuum of learning. This study gains momentum from the findings of a previous study conducted by GF.9\n\nIn order to meet the needs identified in curriculum development, this study used a Delphi method approach to identify teaching strategies that aim to develop interprofessional competencies in undergraduate health care students at the University of the Western Cape. The study goes beyond not only identifying teaching strategies, but also considers assessment strategies that could be used to develop interprofessional education (IPE) curricula.\n\n\nMethods\n\nThe study was approved by the Senate Research Committee of the University of the Western Cape, South Africa (registration number: 14/9/25). Participation in the study was voluntary and written informed consent was obtained from participants beforehand to use their data for analysis and publication. The data were collected and processed anonymously.\n\nDenzin and Lincoln states that qualitative research offers methodological tools with which to understand the deeper meanings associated with multifaceted phenomena and processes in practice.15 Besides the traditional approaches to qualitative inquiry, such as grounded theory, phenomenology, constructivist inquiry, and narrative inquiry, the Delphi method is an additional approach not often highlighted in the literature. The Delphi method is a logical approach based on the philosophical assumptions of philosopher and educator John Dewey, who believed that social science research should directly relay and inform everyday practice and decision-making.16 According to Birdsall, the Delphi method stresses structured anonymous communication between the authors and the expert on a certain topic with the aim of reaching consensus in the areas of policy, practice, or organizational decision-making.17,18 The Delphi method used in research usually involves approximately three rounds of surveys that are distributed to a panel of experts, with each round being informed by responses to the previous one. The Delphi process can be continuously repeated until consensus is reached. In this study the Delphi method was used to reach consensus on the most appropriate activities and assessment methods to use in an interprofessional curriculum, that would assist in instilling interprofessional core competencies in undergraduate health care students.\n\nSelection of the appropriate participants is regarded as one of the most important phases in the entire Delphi process, as it directly impacts on the quality of the results produced.19–21 Since the Delphi technique concentrates on prompting expert views over a short period of time, the selection of participants is usually reliant on the disciplinary areas of knowledge and skills required by the specific issue at hand.22 As interprofessional education is a relatively developing area in South Africa, and as such it was initially difficult to identify local experts in the field. The authors aimed to recruit 15 and 20 participants and names were garnered from the initial experts identified, so as to include as diverse a group of experts as possible. The authors approached 95 experts which were more participants than required, in the event that if some were not available, there would be enough participants for the study. Following this process, the participants made up a group of 29 participants. The first phase of the study had 11 out of the 29 invited participants positively respond to participate in the study, yielding an initial response rate of 37.93%. Ludwig15 indicates that “the majority of Delphi studies have used between 15 and 20 respondents”. Based on this, a decision to expand the expert panel base and invite more participants with a target of achieving a positive response of between 15 and 20 was made and executed. Forty potential participants from the African Interprofessional Network (AfrIPEN) were invited to participate in the study. Eight gave a positive response, making a total of 19 participants. The demographics of 17 of these participants can be found in Table 1. In round one, all 19 experts participated and in round two, 16 of the experts completed the questionnaires in the Delphi process.\n\nThe experts in this group were initially contacted via e-mail and came from various organisations, both local and international. International organisations included the Centre for the Advancement of Interprofessional Education (CAIPE), UK, University of Missouri, U. S, Suez Canal University, Egypt, University of Cairo, Egypt, University of North Carolina, U. S, University of North Texas, U. S, Curtin University, Australia, University for Development Studies, Ghana, and the University of Sudan. South African institutions included Stellenbosch University, University of the Western Cape, University of Cape Town, University of Pretoria, University of KwaZulu-Natal, University of the Free State, and Psych Care in Pietermaritzburg.\n\nThe authors obtained contact information of potential participants from publications of experts in high impact journals, organisations/networks like the Africa Interprofessional Education and Collaborative Practice Network (AfIN) and African Interprofessional Network (AfrIPEN). Participants contacted, also identified other experts and made their contact details available to the authors. All the identified participants and experts in the field of IPE received an invitation letter via e-mail, containing information regarding this study and a request for their assistance as an expert in the field of IPE (see Extended data49). A consent form was attached to the e-mail, which needed to be completed and returned to the authors, should they agree to participate in the study (see Extended data49). Once all the consent forms had been received, the participants were sent a link to begin the Delphi process, by completing an online questionnaire in Google Forms. The first section of the questionnaire included a demographic aspect whereby participants had to indicate their profession, years of experience in IPE, year level of student engagement in IPE and the average number of students engaged in IPE per annum. The Psychology Ethics Committee of the University of Aberdeen posits that it is good practice to assign a numerical reference to participants in research studies for the purposes of anonymity.23 This was particularly necessary in this study in order to track participants’ replies and verify their responses during the next round of the Delphi study. The questionnaire was based on the six interprofessional core competencies identified by the Canadian Interprofessional Health Collaborative (CIHC), whereby participants were asked to identify activities and methods of evaluation for each competency domain.24 The questionnaire was sent online, which allowed participants to complete it at a time and space in which they were comfortable. The authors enabled settings in Google Forms to be notified via e-mail when questionnaires had been completed by participants, according to their allocated participant number and, through this method, the panel of experts could keep track of the total number of completed questionnaires.\n\nPrior to the Delphi study, the authors presented the two competency documents (CIHC & Interprofessional Education Collaborative Expert Panel) to faculty who collectively recommended the use of the six competency domains outlined by the CIHC.4,24 The participants in the Delphi study had to review the combined six competencies listed by the CIHC and the two additional competencies suggested by an Interprofessional Education Collaborative Expert Panel. For the purposes of this study, the focus is primarily on the six competencies listed by the CIHC, together with the additional core competencies of the Interprofessional Education Collaborative Expert Panel, i.e., values/ethics for interprofessional practice and roles, as well as responsibilities for the sake of comprehension. Round one required participants to list as many activities as possible, to instil each of the eight core competencies into undergraduate students. While listing activities, they had to think of different assessments that could be used to evaluate the different competencies.\n\nDuring round two, the authors compiled a second questionnaire whereby participants had to rate the activities and assessment practices most favourable to instil IPE core competencies as presented in round one. The scale of reply extended from one to five, ranging from strongly agree to strongly disagree. The most common activity types and assessment methods were selected by the authors from round one. Items were considered as ‘common’ where three or more participants made the same comment. The participants were given a space on the questionnaire to make any further comments should they feel that the items list was not appropriate or in alignment with comments they had made previously. Participants had to state whether they agreed with the listed assessments and activities by clearly stating “yes” or “no”. Since there were no objections and no comments made indicating the inappropriateness of the listed items, the authors concluded that consensus was reached at the completion of round two. This decision was communicated to all participants, in addition to giving participants a final opportunity to dispute the decision, of which there were none.\n\n\nAnalysis\n\nThe questionnaires in the Delphi process included both qualitative and quantitative aspects. Hsu et al., emphasise that researchers need to find a suitable process to deal with the qualitative information collected.22 In this study, the qualitative data in the form of comments was read together with suggested activities and assessment practices to further understand the reasons for listed items.\n\nFor each round in the Delphi study, experts were invited to respond to scale each statement on a Likert-type scale with an option to comment on each statement as desired and finally ranking the statements in the order of importance.25 Quantitative analysis of the Delphi study included calculations of the percentage response rates; percentages for each level of agreement for each statement to compensate for varying response rates; the median, range and their associated group rankings using the importance ratings; mean (SD) and their associated group rankings using the importance ratings. The final results from the Delphi study were reported in percentages to reflect the rate of agreement between experts. Two nominal categories were formed to report the data from the Likert scale used by the authors. Strongly Agree and Agree were combined and Disagree and Strongly Disagree were combined for the purposes of reporting the findings. Green14 suggests that at least 70% of the Delphi participants need to rate three points or higher on a four-point Likert-type scale to reach consensus on subject matter.\n\nHasson et al., states that the Delphi technique is based upon the assumption of safety in numbers (i.e. many experts are less likely to arrive at a wrong decision than a single person).26 Choices are then strengthened by logical argument in which assumptions are confronted, thus helping to increase validity. Threats to validity arise primarily from pressures for convergence of predictions which challenges the Delphi's forecasting capability. However, the use of experts on a particular topic, can assist to increase the content validity of the Delphi technique by using successive rounds of the questionnaire which increases the concurrent validity.26\n\nTrustworthiness of the data was ensured by using Guba’s11 four criteria of trustworthiness:\n\ni) Credibility\n\nThe authors adopted appropriate, well-recognised research methods, which were familiar to the culture of the participating institution and used random sampling of individuals who served as participants in the study. Triangulation was done by the use of different methods, and different selected participants were used for different phases of the research study in varying contexts. Detailed descriptions were given of the background to the study and member-checks of data collected were done in the Delphi study by allocating numbers to participants and getting them to confirm data.\n\nii) Transferability\n\nThe authors provided background data in the study to establish the specific context and gave a detailed description of the phenomenon in question, to allow comparisons to be made with other/similar institutions.\n\niii) Dependability\n\nThe different methods used in this study allowed for overlap and integration in order to develop an IPE model. In-depth methodological description was given in chapter two, which allows this study to be repeated.\n\niv) Confirmability\n\nResearcher bias was reduced through triangulation of the data and all assumptions and beliefs of the authors were outlined in each chapter. Shortcomings in the methodology of the study and their likely effects are listed in the final chapter of the study as limitations and an in-depth methodological description is provided so as to allow integrity of the research results which can be scrutinised by experts in the field.\n\n\nResults\n\nSuggested activities and assessments made by the participants for the IPE core competencies were ranked and captured accordingly (see Table 2).48\n\nFourteen of the experts participated in this round. The suggestions given for activities and assessment strategies that were common to the majority of participants, were summarised and sent back to the participants for confirmation in the form of round two.48 Participants were requested to rate common suggestions on activities and assessments given as Likert items (see Table 3 and Table 4) and make necessary comments should there be any discrepancies.\n\n\nDiscussion\n\nConsidering the above-mentioned assortment of activities, it is evident that similar activities can be used to instil more than one competency, for example, case studies that mention interprofessional communication, patient/client/family/community-centred care, role clarification, interprofessional conflict resolution and values/ethics for interprofessional practice. Another example is role play, which can be used to develop the core competencies of role clarification, collaborative leadership, interprofessional conflict resolution, and values/ethics for interprofessional practice. However, when considering such overlap, it could appear repetitive and confusing in nature when designing new IPE activities and curricula. Barr et al. provide some guidance on how to classify different learning activities that are frequently used in IPE.27 They state that using different methods in combination with each other can be very advantageous for students. The classification is as follows and the results are discussed accordingly:\n\ni) Exchange-based learning, e.g. case studies and debates\n\nii) Action-based learning, e.g. workshops, problem-based learning, collaborative enquiry and continuous quality improvement\n\niii) Observation-based learning, e.g. joint visits to a patient by students from different professions, shadowing another profession\n\niv) Simulation-based learning, e.g. role-play, games, skills labs and experiential groups\n\nv) Practice-based, e.g. co-location across professions for placements, out-posting to another profession and interprofessional training wards\n\nvi) E-learning, e.g. reusable learning objects relating to the above\n\nvii) Blended learning, e.g. combining e-learning with face-to-face learning\n\nviii) Received or didactic learning, e.g. lectures.\n\nThe following main activities that were highlighted by the expert panel and were common to most of the IPE core competencies; case studies, joint clinical placements, simulations, role plays and workshops/discussions.\n\nCase studies can be considered as a problem-based learning approach and classified under exchange-based learning, according to Barr et al.27 Bonney highlights several advantages of using case studies as a teaching strategy.28 Firstly, case studies improve the development of the higher levels of Bloom’s taxonomy of cognitive learning,29 which moves beyond not only recalling knowledge, but includes analysis, evaluation, and application. Secondly, case studies facilitate interdisciplinary learning and can be used to facilitate connections between specific theory and real-world societal issues and applications. Case studies have the ability to increase student motivation to participate in class activities, which promotes learning and improves performance on assessments.\n\nStudents in groups can be presented with a well-structured problem or case study in which they have to work collaboratively in a once-off session of a week or longer duration, depending on the outcomes of the session. This would encourage active learning among team members. A case study lends itself to being open-ended; it allows for realistic problems to be used to stimulate interdisciplinary discussions; promotes critical thinking, learning and participation among students, especially in terms of their ability to view an issue from multiple perspectives and to grasp the practical application of core course concepts.30\n\nWorkshops demonstrate modern principles of teaching such as active engagement of the learners. They provide opportunities for the interaction that enables the teachers to connect the material to the context of the learners. They provide an opportunity for group interaction, which is important for trainees who are becoming increasingly isolated in their work.31 When planning workshops, it is suggested that student preparation and attendance should be a requirement, allowing for a greater success of the workshop.32\n\nThere are two activities classified under simulation-based activities, role plays and simulations. Simulations provide students of all professions a safe space to interact with each other collaboratively, as well as opportunities for a novice’s eventual transition to becoming an expert. Simulated activities provide students with an opportunity to explore and appreciate the roles of other health professionals. Fowler-Durham and Alden confirm that simulation intends to mimic reality whilst offering a skills-based clinical experience in a safe and secure setting.33 Hovancsek describes the aim of simulation as ‘to replicate some or nearly all of the essential aspects of a clinical situation so that the situation may be more readily understood and managed when it occurs in reality in clinical practice’.34 Russell et al., state that role plays and simulations function as learning tools for teams and groups or individuals, as they can either engage with each other online or face to face.35\n\nJoint clinical placements are a vital part of undergraduate education, allowing students to transform theory into practice by engaging in ‘real-life’ experiences, to strengthen the academic programme content covered at the institution. Koh warns that students who are unable to link theory and practice could possibly be left ‘floundering, lacking in confidence and disenchanted, with some being forced to leave’.36 The core element of a clinical placement is that learning occurs by doing, since problems associated with clients/patients are placed in context and critical thinking can be developed.37\n\nThe main suggestions given by the expert panel on assessment methods aligned to the suggested activities are portfolios, reflection and the development of appropriate rubrics which will be discussed below.\n\nPortfolios are ideal as an assessment tool as they allow for critical analysis of their contents, which reflects on a particular student/group/community. They can therefore be considered as multipurpose instruments, since they can be used for assessments, monitoring and planning, reflection, learning, and for personal development.38 Portfolios are known to stimulate reflection, as students are often required to look back on work they have done and analyse what they have or have not achieved and supply reasons for this. Portfolios are often compiled over a long period of time to allow a sufficient interval in which to collect information and to reflect on the knowledge that has been gained from these experiences. Brown defines a portfolio as ‘A private collection of evidence, which demonstrates the continuing acquisition of skills, knowledge, attitudes, understanding and achievements. It is both retrospective and prospective, as well as reflecting the current stage of development and activity of the individual’.39 Students can sort the evidence in their portfolios into sections corresponding to the different competencies to be assessed and use captions to explain what the evidence shows about a specific competency, since many medical curricula are based on competency criteria.38\n\nSandars states that many assessments include ‘levels of reflection’ and that this hierarchical model is based on the notion of depth of reflection.40 Superficial reflection is considered to occur when there is only a report of events, but deeper reflection includes a ‘stepping back’ from events and actions with evidence of the encounter and possible change to current views and perceptions. This deeper level is equivalent to ‘transformative learning’ taking place. Reflection can be considered as a purposeful critical analysis of knowledge and experience in order to achieve a deeper meaning and understanding of a specific body of information. Reflection cannot be seen in isolation from reflective learning and reflective practice.40 In a study done by Morison et al. on reflection, students felt that learning together in both lectures and on clinical placement allowed them to gain optimum understanding on their own and others’ roles and that the real-world experience helped them to appreciate the importance of teamwork and communication skills.41 Mann et al., confirm that professional competencies can be assessed through reflection and that different levels of reflection should be established for each year level.42\n\nThe third assessment method highlighted by the expert panel is the use of rubrics. Rubrics are a good indicator to students of what aspects of their performance will take priority and how marks/percentages will be allocated to specific tasks for assessment purposes. The use of rubrics in assessments offers a means to provide the desired validity in assessing complex competencies, without forfeiting the need for reliability.43,44 When designing rubrics, Reddy strongly suggests that assessors ensure that the scoring criteria reflect the desired core competencies that would suggest success in curriculum design and practice.45 The scoring/rating of rubrics are descriptive scoring schemes that are developed by educators or others (clinicians/supervisors/peers) to guide the analysis of written work or practical work in terms of a process towards students’ efforts.46\n\nConsidering how the activities above can instil core competency development and the suggestions given on how to assess the activities, it cannot be successfully adopted without the suggestions given by experts. For novice higher education institutions that are wanting to design an IPE curriculum, the experts in the field of interprofessional education suggested the application of principles such as treating all the health professions as equals, showing mutual respect, valuing differences, working towards common goals, commitment from the faculty including its leadership, alignment of timetables which includes shared curriculum and assessment practices, to name but a few. Core competency development has been hugely guided from international influences but cannot be applied to all contexts without adaptations.\n\n\nConclusion\n\nThis study used a Delphi approach to identify teaching and assessment strategies that aim to develop interprofessional competencies in undergraduate health care students. Consensus has been reached by an expert panel on learning activities and assessment methods that instill the development of interprofessional core competencies. These identified strategies will form a crucial aspect in developing an IPE curriculum, especially in a South African context. The learning outcomes in such an IPE curriculum need to be clearly outlined and linked to each respective year level of training for health professions training at tertiary institutions. There is growing evidence that intensive approaches to learning are more likely to be connected with higher quality learning outcomes.47 The development of an IPE model that incorporates a curriculum as described above will allow for flexible application of these learning outcomes that are both challenging and reflective of the cognitive level of learning across the learning continuum.\n\nAlthough the number of experts who were classified as the participants for the study fell into the normal range for Delphi studies, a larger number of participants would have yielded a more enriched data set. The first round of the Delphi study took almost a year to complete as the authors had to send out monthly reminders to participants who had consented to participate in the study.\n\nIPE is an emerging field and the literature is constantly growing with more and more experienced academics and practitioners emerging. In light of the COVID-19 pandemic, many higher education institutions have resorted to online learning and activities for IPE. Thus, it would be worthwhile to do a follow-up survey to get feedback on these activities and assessment strategies which can be added to this study.\n\n\nData availability\n\nFigshare: Consensus on activities and assessment methods that instil interprofessional core competencies (data sets), https://doi.org/10.6084/m9.figshare.17134679.v1.48\n\nThis project contains the following underlying data:\n\n• Delphi Responses – Round 1.pdf (Identification of activities and assessments that develop interprofessional core competencies)\n\n• Delphi Confirmation – Round 2.pdf (Confirmation of activities and assessment strategies that develop interprofessional core competencies)\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: Consensus on activities and assessment methods that instil interprofessional core competencies (appendices), https://doi.org/10.6084/m9.figshare.18771038.v1.49\n\nThis project contains the following extended data:\n\n• Invitation Letter.pdf\n\n• Delphi questionnaire – Round 1.pdf\n\n• Delphi questionnaire – Round 2.pdf\n\n• Consent form.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nMcKean SCW, Budnitz TL, Dressler DD, et al.: How to use The Core Competencies in Hospital Medicine: a framework for curriculum development. 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Publisher Full Text\n\nBirdsall I: It seemed like a good idea at the time: The forces affecting implementation of strategies for an information technology project in the Department of Defense. Virginia Tech. 2003; 1–242. Reference Source\n\nDalkey N, Helmer O: An Experimental Application of the Delphi Method to the Use of Experts Author (s): Norman Dalkey and Olaf Helmer Published by: INFORMS Stable. Accessed: 27-04-2016 21: 16 UTC. 1963;9(3):458–67. Reference Source\n\nJudd RC: Delphi decision methods in higher education administration. Technol Forecast Soc Change. 1972; 4(2): 173–186. Publisher Full Text\n\nTaylor RE, Judd LL: Delphi method applied to tourism. Delphi method Appl to Tour. 1989; 95–98.\n\nJacobs JM: Essential assessment criteria for physical education teacher education programs: A Delphi study.1996.\n\nHsu CC, Sandford BA: The Delphi technique: Making sense of consensus. Pract. Assess. Res. Eval. 2007; 12(10): 1–8.\n\nPEC PEC: Psychology Ethics General Guide. Univ. Aberdeen; 2017 [cited 2017 Aug 21]. Reference Source\n\nOrchard C, Bainbridge L, Bassendowski S, et al.: A National Interprofessional Competency Framework. Vancouver: Canadian Interprofessional Health Collaborative; 2010.\n\nHoley EA, Feeley JL, Dixon J, et al.: An exploration of the use of simple statistics to measure consensus and stability in Delphi studies. BMC Med. Res. Methodol. 2007; 7: 1–10.\n\nHasson F, Keeney S, McKenna H: Research guidelines for the Delphi survey technique. J. Adv. Nurs. 2000; 32(4): 1008–1015. PubMed Abstract\n\nBarr H, Koppel I, Reeves S, et al.: Effective Interprofessional Education. Oxford, UK: Blackwell Publishing Ltd; 2005. Publisher Full Text\n\nBonney KM: Case Study Teaching Method Improves Student Performance and Perceptions of Learning Gains. J Microbiol Biol Educ. 2015; 16(1): 21–28. PubMed Abstract \| Publisher Full Text\n\nChurches A: Wiki Editting Rubric - Bloom’s Digital Taxonomy. Educ Origami. 2007; 19(12/07): 1–44.\n\nYadav A, Lundeberg M, DeSchryver M, et al.: Teaching Science with Case Studies: A National Survey of Faculty Perceptions of the Benefits and Challenges of Using Cases. J. Coll. Sci. Teach. 2007; 37(1): 34–38.\n\nEison J, Stevens E: Faculty development workshops and institutes. Teaching improvement practices: Successful strategies for higher education.1995; 206–27.\n\nVanKuiken DM, Schaefer JK, Flaum Hall M, et al.: Integrating interprofessional education into the curriculum: Challenges and solutions for a university without a medical center. J. Interprofessional Educ. Pract. 2016; 2: 5–11. Publisher Full Text\n\nDurham CF, Alden KR: Enhancing Patient Safety in Nursing Education Through Patient Simulation. Patient Saf Qual An Evidence-Based Handb Nurses. 2008. Reference Source\n\nHovancsek MT: Using simulation in nursing education. Simul Nurs Educ From Conceptualization to Eval. 2007; 1(9).\n\nRussell C, Shepherd J: Online role-play environments for higher education. Br. J. Educ. Technol. 2010; 41(6): 992–1002. Publisher Full Text\n\nKoh L: Practice-based teaching and nurse education. Nurs. Stand. 2002; 16(19): 38–42. Publisher Full Text\n\nNolan CA: Learning on clinical placement: The experience of six Australian student nurses. Nurse Educ. Today. 1998; 18(8): 622–629. PubMed Abstract \| Publisher Full Text\n\nVan Tartwijk J, Driessen EW: Portfolios for assessment and learning: AMEE Guide no. 45. Med. Teach. 2009; 31(9): 790–801. PubMed Abstract \| Publisher Full Text\n\nBrown RA: Portfolio Development and Profiling for Nurses. White Cross, Lancaster: Quay Books: a division of M.A. Healthcare Ltd; 2nd ed.1995.\n\nSandars J: The use of reflection in medical education: AMEE Guide No. 44. Med. Teach. 2009; 31(8): 685–695. PubMed Abstract \| Publisher Full Text\n\nMorison S, Jenkins J: Sustained effects of interprofessional shared learning on student attitudes to communication and team working depend on shared learning opportunities on clinical placement as well as in the classroom. Med. Teach. 2007; 29(5): 450–456. PubMed Abstract \| Publisher Full Text\n\nMann K, Gordon J, MacLeod A: Reflection and reflective practice in health professions education: A systematic review. Adv. Health Sci. Educ. 2009; 14(4): 595–621. PubMed Abstract \| Publisher Full Text\n\nMorrison GR, Ross SM: Evaluating Technology-Based Processes and Products.1998; (74): 69–77.\n\nWiggins G: Educative Assessment. Designing Assessments To Inform and Improve Student Performance. 350 Sansome Street, San Francisco, CA 94104: Jossey-Bass Publishers; 1998.\n\nReddy MY: Design and development of rubrics to improve assessment outcomes: A pilot study in a Master’s level business program in India. Qual. Assur. Educ. 2011; 19(1): 84–104. Publisher Full Text\n\nBrookhart SM: Teaching about grading and communicating assessment results. Pap. Present Annu. Meet. Natl. Counc. Meas. Educ. 1998.\n\nProsser M, Trigwell K: Understanding Learning And Teaching: The Experience in Higher Education. McGraw-Hill Education; 1999.\n\nFilies GC, Frantz JM: Consensus on activities and assessment methods that instil interprofessional core competencies (data sets).2021. Reference Source\n\nFilies G, Frantz J: Consensus on activities and assessment methods that instil interprofessional core competencies (appendices). figshare. Dataset. 2022. Publisher Full Text" }	[ { "id": "121501", "date": "04 Feb 2022", "name": "Champion Nyoni", "expertise": [ "Reviewer Expertise Health Professions Education and Nursing 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\nThank you for the opportunity to review the submitted manuscript titled “Towards an interprofessional competency-based model in South Africa: A Delphi Study”. In this study, the authors engage with experts in identifying activities that could be used to facilitate interprofessional education. I will present my comments based on the guidelines for reviewers according to the journal.\nIs the work clearly and accurately presented and does it cite the current literature? Several issues need to be addressed in this work to enhance clarity and accuracy. The abstract should be re-written so that it aligns the purpose of the work and the intended outcomes. The premise of this paper is based on a systematic review conducted in 2017 regarding teaching activities for IPE, where the authors go on to say- from that systematic review, they did not find any studies in South Africa that had been conducted in relation to the subject. Several IPE activities have been reported from that time that reflect activities to instil IPE competences. I refer the authors to a special issue by the Journal of Interprofessional Care (Vol. 33, issue 3 [pages 373-342], 2019) that focused on IPE in Southern Africa. Most of the authors in that special issue were from South Africa. This is an essential aspect of the literature that should have been consulted in the crafting of this work. Authors must review the latest work in the field and position their argument based on such work. I also noticed a source from the seventies, is this seminal work?\nIs the study design appropriate and does the work have academic merit? The chosen design aligns with the purpose of the work. However, the execution of this design needs to be revised. The authors set off their discussion of the design from a qualitative approach which does not align or fit with the paradigm nested within a Delphi technique. The authors must explain the criteria of choosing the experts in this study, what qualifications did they have, what was their experience in relation to IPE. There is a section of “trustworthiness” that appears to have been extracted from a thesis submitted at the University of the Western Cape – word for word. In addition, the elements referred to in this section do not align with the design and the paper, for example the authors under the heading “dependability” mention that ‘methodological descriptions are given in Chapter two’. There is no chapter two in this paper. The authors need to be aware of self-plagiarism.\nAre sufficient details of methods and analysis provided to allow replication by others? The method of calculating consensus is not explicit. There are several authors that have argued that there is a difference between consensus and agreement and these concepts apply different analysis approaches. In this study, the authors use these concepts interchangeably and this is leading to some lack of clarity for possible replication. See Meijering et al. (2013) Quantifying the development of agreement among experts in Delphi studies. Technological Forecasting and Social Change 80(8) pg 1607-1614. The methods must be explicitly described to enhance transferability and based on sound literature.\nIf applicable, is the statistical analysis and its interpretation appropriate? The statistical analysis needed to be reworked. It is not clear what the authors used to determine the consensus. There is mention of 70% as cut-off but then this is not clear how this was applied. The results still show some areas were there was not 70% agreement. Some of the results in the rating scale do not align with the activities for example, table 3 has 5 activities and 7 rows of results. This does not align- making it difficult to trust these results.\nAre all the sources’ data underlying the results available to ensure full reproducibility? Yes, there have been sufficiently provided.\nAre the conclusions drawn adequately support by the results? The discussion of the study does not align with the overall purpose of the study. At this stage, it is not clear how the sampled activities methods were included for discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "135432", "date": "04 May 2022", "name": "Kanako Noritake", "expertise": [ "Reviewer Expertise Dental Education", "Interprofessional Education." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for the opportunity to review the submitted manuscript titled “Towards an interprofessional competency-based model in South Africa: A Delphi Study”. In this study, the authors tried to identify teaching strategies and activities that aim to develop interprofessional competencies in undergraduate health care students at the University of the Western Cape, South Africa. I will comment based on the guidelines for reviewers according to the journal.\nIs the work clearly and accurately presented and does it cite the current literature? Although I think it is important to establish an educational program based on the context of each region, there have been so many research reports on IPE all over the world since 2017, and this paper lacks references on the IPE core-competencies. At least, they should check these two guidelines; 1.CORE COMPETENCIES FOR INTERPROFESSIONAL COLLABORATIVE PRACTICE: 2016 UPDATE, 2. INTERPROFESSIONAL EDUCATION GUIDELINES 2017 by CAIPE.1,2 There is a worldwide consensus on strategies for IPE core competencies, so the study should be based on this.\nIs the study design appropriate and does the work have academic merit? Although I am not medical education expert, I don't think the Delphi method is appropriate for the purposes of this study. Since the purpose of this study is to identify educational strategies and activities aimed at developing interprofessional competencies among undergraduate medical students at the University of the Western Cape in the Republic of South Africa, it is necessary to examine the various IPE that has been conducted elsewhere and then consider the competencies required of students at the University of the Western Cape. It would be necessary to do so, but it does not appear from the methods of this journal that such a process has been adequately undertaken.\n\nAre sufficient details of methods and analysis provided to allow replication by others? They mentioned \"Round one required participants to list as many activities as possible, to instil each of the eight core competencies into undergraduate students.\" (P5L20-22). Can this method be called the Delphi method?\nThey also mentioned that \"For each round in the Delphi study, experts were invited to respond to scale each statement on a Likert-type scale with an option to comment on each statement as desired and finally ranking the statements in the order of importance.\". I think experts ranked the Likert-scale only during round two, or they ranked twice? I am confused.\nIf applicable, is the statistical analysis and its interpretation appropriate? I feel that the method of analysis of the Delphi method data in this study is not valid. If they had combined strongly agree and one agree, and disagree and strongly disagree, it would no longer be different from a three or more level Likert.\nAre all the sources’ data underlying the results available to ensure full reproducibility? Yes, I think so.\nAre the conclusions drawn adequately supported by the results? I think the aim of this study and the conclusion doesn't match.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/11-116
https://f1000research.com/articles/11-115/v1	28 Jan 22	{ "type": "Research Article", "title": "Knowledge, attitude, and practices associated with avian influenza among undergraduate university students of East Java Indonesia: A cross-sectional survey", "authors": [ "Saifur Rehman", "Fedik Abdul Rantam", "Khadija Batool", "Attaur Rahman", "Mustofa Helmi Effendi", "Jola Rahmahani", "Muhsin Jamal", "Saifur Rehman", "Fedik Abdul Rantam", "Khadija Batool", "Attaur Rahman", "Jola Rahmahani", "Muhsin Jamal" ], "abstract": "Background: Several public health strategic actions are required for effective avian influenza (AI) prevention and control, as well as the development of a communication plan to keep undergraduate students sufficiently informed on how to avoid or reduce exposure. The aim of the survey was to measure the level of knowledge, attitudes and practices (KAPs) toward AI among undergraduate university students in East Java, Indonesia, and observe the correlation between KAPs and the factors associated with the control and prevention of AI. Methods: A cross-sectional survey was conducted among undergraduate students to collect information about AI-related KAPs. Students were selected from three faculties of Universitas Airlangga Surabaya Indonesia (Faculty of Veterinary Medicine, Faculty of Fisheries and Marine, and Faculty of Science and Technology). Students voluntarily responded to a pre-designed questionnaire.\nResults: A total of 425 students (222 female; and 203 male), of ages ranging from 18 years (n=240) to 20-30 years (n=185), responded to the survey. This cohort consisted of 157 students from the Faculty of Fisheries and Marine, 149 from the Faculty of Veterinary Medicine, and 119 from the Faculty of Science and Technology. The results indicated that appropriate knowledge was obtained by 76.94% of students; significantly higher levels were seen in Faculty of Veterinary Medicine students as compared to the other two faculties (p<0.05). 72.89% of students documented positive attitudes; veterinary medicine students had significantly more positive attitudes than other faculties (p<0.05). Proactive behaviors were observed in 56.90% of students. The aggregate scores for KAPs were 6.93 ± 0.77 (range: 0-9) for knowledge, 7.6 ± 1.25 (range: 0-10) for attitude, and 9.1 ± 1.5 (range: 0-12) for practice.", "keywords": [ "Avian Influenza", "Knowledge", "Attitude", "Practices", "Public Health", "Undergraduates" ], "content": "Introduction\n\nAvian influenza (AI) is a highly contagious viral zoonotic disease, and has become a serious public health concern in the last two decades as a result of a significant increase in the interspecies transmission of AI viruses from diseased birds to humans1,2. Since the first laboratory-confirmed case was reported in Hong Kong in 1997, there have been 860 confirmed human infections with the avian H5N1 virus, resulting in 454 deaths worldwide1. To date, only the H5 and H7 AI subtypes have been associated with naturally occurring, highly pathogenic AI A (HPAI) viruses that cause acute clinical disease in chickens, ducks, turkeys, and other economically significant birds. The majority of AI viruses of the subtypes H5 and H7 identified from birds, have been found to be low-pathogenic to poultry. All H5 and H7 viruses have been categorized as Notifiable Avian Influenza (NAI) viruses, because there is a possibility for lentogenic H5 or H7 viruses to become velogenic through mutation. Humans, rats and mice, weasels and ferrets, pigs, cats, tigers, and dogs have all been found to be carriers of the virus3. In various countries, including Vietnam, Indonesia, Thailand, China, Cambodia, and, more recently, Turkey and Iraq, AI strains have been found in birds, including wild and commercial poultry4.\n\nIn Indonesia, the majority of human cases of HPAI H5N1 have occurred in the western part of the main Island of Java. Although minimal, non-sustained human-to-human transmission has undoubtedly occurred, and the transmission was linked to poultry exposure, such as direct or close contact with infected or dead birds or visiting a live poultry market1,5–10. Late presentation to health care and hospitalization, delayed clinical detection of AI and delayed antiviral therapy have all been related to increased mortality from HPAI H5N1 virus infection in Indonesia7,11–13. Knowledge, attitude, and practices (KAPs) are one of the most important factors in controlling and preventing the spread of certain diseases or infections to public health14–16. In the past few years, infected countries like Thailand, China, Italy, Turkey, and Afghanistan have been studying KAPs towards AI. Workers in the poultry industry had the highest risk of contracting AI. A study of Italian poultry workers was undertaken to assess their KAPs toward AI, and it was discovered that workers' awareness of transmission and prevention measures might be enhanced because of their close contact with poultry17. A KAP questionnaire is a comprehensive survey conducted on a specific population to determine a population's knowledge (K), attitudes (A), and practices (P) on a particular topic. In most KAPs studies, an interviewer uses a standardized, structured questionnaire to obtain data orally18. An earlier survey was carried out in Thailand among the rural community on KAPs towards AI, revealing that a public education campaign was beneficial in promoting AI disease prevention methods to the public19. However, there is a paucity of research on the KAPs towards AI in Indonesia. Considering the pandemic classification of AI and its critical consequences in terms of economic losses to the poultry industry and serious deleterious effects on public health, a survey was undertaken among undergraduate university students at a public sector university, to explore the benefits of adopting a KAP strategic model in controlling and preventing AI in Indonesia. The aims of the survey was to measure the level of KAPs towards avian flu among the students, and observe the correlation between KAPs and the factors associated with control and prevention of AI. The findings will significantly help to understand the basic knowledge of AI, its clinical manifestation, pathogenesis, routes of transmission, broad range of hosts, and pandemic nature of the virus, acquired by undergraduate students in particular, and Indonesians in general.\n\n\nMethods\n\nThe respondents of this survey were the undergraduate students (from the Faculty of Veterinary Medicine, Faculty of Fisheries and Marine, and Faculty of Science and Technology) of the University of Airlangga Surabaya Indonesia. The study was conducted from April 23 to May 20, 2021, during the peak of the COVID-19 pandemic. After obtaining ethical approval from the Faculty of Veterinary Medicine Research Ethics Committee, the respondents were interacted with through face-to-face meetings to respond to the study questionnaire. This measure was taken due to the practical nature of this study that required self-administration for rapid data collection. The advantage of this method includes handling missing values, consistency of data findings, transparency of personal interest and the study framework as per literature guidance20.\n\nWe contacted 425 undergraduate students from three different faculties, of Universitas Airlangga of ages ranging between 18 to 30 years to get the questionnaire filled. Informed consent was obtained from participants and anonymity of personal information was also considered. The sample size was determined based on a 5% precision level of faculty population.\n\nThese faculties were chosen because most students from the Faculty of Veterinary Medicine carry out research related to zoonotic diseases; while few students from other faculty carry out research on zoonotic diseases, they are part of their curriculum, such as AI, salmonella, and klebsiella. All participants were informed about the survey before filling the questionnaire with the following statements “All participation in this study is completely voluntary, if you decide not to participate there will not be any negative consequences. Please be aware that if you decided to participate, you may stop participating any time and you may decide not to answer any specific question. The aim of the survey is to measure the level of KAPs toward avian flu among the students. You will receive no direct benefits from participating in this research study. However, your responses may help us learn more about KAPs of avian influenza”.\n\nThe questionnaire contained four demographic characteristics (age, gender, hometown, and name of faculty); KAP parameters were a set of nine AI knowledge variables (avian influenza is a contagious infection, avian influenza is caused by highly pathogenic influenza A [H5N1] virus, avian influenza is similar to swine influenza regarding their signs and symptoms [transmission] animal-to-animal, animal-to-human, human-to-human (risk group) poultry workers, butchers, veterinarians); five AI attitude variables (washing hands before eating and after touching raw poultry meat, using gloves to touch raw poultry meat, preparing raw poultry and other foods using different knives, and cleaning the cutting boards after preparing raw poultry meat); and six AI practice variables (washing hands with soap before and after eating, covering nose when sneezing and coughing, wearing a surgical mask and consultation with the doctor promptly in case of suspected contamination). All questions were developed based on a published questionnaire from a study on Italian poultry workers21. For this KAP questionnaire, a standard scoring method was used encompassing all KAP sections. In the knowledge section, correct answers were scored 1 point and 0 points were given for incorrect responses, while in the attitude section positive options obtained 2 points, neutral options obtained one point, while negative options get zero points. Similarly, in the practice portion, 2 points were awarded for proactive actions, 1 point was given for neutral actions, and 0 points for passive actions.\n\nThe data collected through a standard KAP questionnaire module were subjected to statistical analysis by using the SPSS 25.0 software package. Both descriptive and inferential statistical tests (Chi-square) were applied to compare categorical response variables and ratios, and to assess their statistical significance (p<0.05).\n\n\nResults\n\nAll undergraduate students (n=425) were selected from three different faculties of Universitas Airlangga Surabaya Indonesia. Among them, 52.2% (222 out of 425) were females and 47.8% (203 out of 425) were males. A total of 35.05% (149 out of 425) of the respondents were from the Faculty of Veterinay Medicine, 36.94% (157 out of 425) from the Faculty of Fisheries and Marine, and 28% (119 out of 425) students from the Faculty of Science and Technology. A 56.5% fraction (240 out of 425) of participants were <20 years old while 43.5% (185 out of 43.5%) were aged between 20–30 years.\n\nAI-associated knowledge was assessed by five questions. Each question-and-answer is described with graded scores in Table 1. Among the total 3,825 answers, 2,943 (76.94%) were correct. Significantly higher scores were found in Faculty of Veterinary Medicine students for K1, K5, K6, and K8 as compared to those from the Faculty of Fisheries and Marine and the Faculty of Science and Technology (p<0.05), but no other statistical significance was found among the groups (Table 2).\n\nTable 2 lists the questions and correct answers for each variable.\n\nThe percentage of correct information between different groups was compared using the Chi-square test.\n\nThere were five categories of AI attitude questions. Each question-and-answer option is presented in detail with scores in Table 3. In total, 1,549 (72.89 %) of the 2,125 responses showed a positive attitude (Table 3). Faculty of Veterinary Medicine students' scores were significantly higher than other faculties for A4 and A5 (p<0.05), while no other variable showed statistically significant differences between faculties (Table 4).\n\nTable 4 lists the questions and correct options for each variable.\n\nThe percentage of positive attitudes between different groups was compared using a Chi-square test.\n\nSix questions were used to assess practices related to AI; each question-and-response option, along with their graded scores, are given in Table 5 and Table 6. Out of 2,550 responses, 1,451 (56.90%) had adopted proactive practices. For questions P1, P4, and P5, veterinary students scored significantly higher than Faculty of Fisheries and Marine students. Students from the Faculty of Science and Technology scored much higher on question P4 than Faculty of Fisheries and Marine students (p<0.05) (Table 6).\n\nTable 6 lists the questions and proactive options for each variable.\n\nThe percentage of proactive options in different groups was compared using the Chi-square test.\n\n\nDiscussion\n\nThe goal of the study was to gather data on AI-related KAPs among undergraduate students from three different faculties of Universitas Airlangga, Indonesia. We found that all the participants were aware of AI. Additionally, public health education was identified as a useful method for preventing and controlling public health emergencies, as well as improving public preparedness in the case of any pandemic22. It encourages the public to gain adequate knowledge to reduce stress and anxiety, develop a positive attitude and keep desirable behaviors under the situation of pandemic23. All of these KAP components have been deemed essential for efficient pandemic prevention and control. This cross-sectional study of 425 undergraduate students indicates that the majority of them were well-informed about AI knowledge, had a positive attitude, and engaged in proactive practices, showing that major public education efforts offered effective health awareness benefits. This finding reflects several previous studies reports on H1N1-related KAPs among university students in South Korea, the United Kingdom (UK), and Hong Kong24–26. Our study revealed that Veterinary Medicine students scored much higher on knowledge than students from other faculties, which could be explained by their exposure to, and training in, clinical medicine and veterinary public health, the concept of One Health and zoonosis. Their obligations and responsibilities as future public veterinary health experts to combat any pandemic are assumed to motivate them to adopt more positive attitudes and proactive behaviors in the event of a public health emergency27. In the attitude section, students from the Faculty of Veterinary Medicine showed a significantly greater positive attitude than the other two faculties, indicating that veterinary students were more aware of the zoonotic importance of AI. This could explain the importance of veterinary education in the current study regarding One Health approaches and the role of the veterinarian in an eco-friendly environment28. Our findings are compatible with the results of previous studies on KAPs towards H1N1 among university students of Hong Kong, South Korea, and the UK24–26.\n\nIn the practice section, students from the Faculty of Veterinary Medicine and Faculty of Science and Technology showed significantly higher scores (p<0.05) as compared to the Faculty of Fisheries and Marine students. These standard practices noted in the current study showed a positive correlation between science and technology and veterinary science courses with education on infectious diseases like AI. Similarly, a prior COVID-19-related KAP study are conducted among undergraduate students in China, revealed that medical and health science students have more proactive practices as compared to other students from different fields of education29.\n\nThere are some limitations to our research that must be noted. First, the nature of the cross-sectional study design limits the ability to draw causal inferences from the observed relationships. Second, our participants were recruited from three faculties within a single university, and attended the university during a pandemic for their research activity, while the majority of students stayed at home at the time of the survey due to the COVID-19 pandemic lockdown.\n\nTo our knowledge, this is the first study of current KAPs related to AI among Indonesian undergraduate students at any university, and it provides useful information regarding public health education and preventative measures in Indonesian universities during any pandemic. Our findings revealed that most of the undergraduate students at the University of Airlangga have a baseline knowledge of AI, although their scores may vary depending on the Faculty. Attitude towards AI showed a discrepancy among the Faculty students. Overall, our findings showed that faculties other than Veterinary Medicine have an impact on students' reactions to AI-related KAPs. In the educational and health sectors, public health education and awareness initiatives are conducted regarding infectious diseases.\n\n\nConclusions\n\nAccording to the findings of this study, the majority of undergraduate students grasped the basic information, had a positive attitude, and showed a proactive behavior toward AI, demonstrating the efficacy and success of current public health education initiatives. However, health and educational institutions should adopt public health trainings, prepare the global population and strengthen their prophylactic measures against any pandemic. The results suggest that the students from the Faculty of Fisheries and the Faculty of Science and Technology should be taken into consideration for future strategic studies of awareness campaigns, public health concerns preparedness, and proactiveness in case of any pandemic.\n\n\nData availability\n\nFigshare: Knowledge, attitude and practices associated with Avian influenza among undergraduate university students of East Java Indonesia: A cross-sectional survey, https://doi.org/10.6084/m9.figshare.16664488.v130\n\nThis project contains the following underlying data:\n\n- Final for spss.xlsx (survey answers data)\n\n- Spss Survey final File.sav\n\nFigshare: Knowledge, attitude and practices associated with Avian influenza among undergraduate university students of East Java Indonesia: A cross-sectional survey, https://doi.org/10.6084/m9.figshare.16664488.v130\n\nThis project contains the following extended data:\n\n- Questionnaire.docx\n\n- Tables.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": "Acknowledgments\n\nThe authors wish to express their sincere gratitude to the staff of the Young Leaders’ Program, Faculty of Veterinary Medicine Universitas Airlangga.\n\n\nReferences\n\nAmin S: Avian Influenza. The Indonesian Experience Paper presented at the Biosafety and Biorisks Conference Lyon, France. 2005.\n\nPeiris JS, de Jong MD, Guan Y: Avian influenza virus (H5N1): a threat to human health. Clin Microbiol Rev. 2007; 20(2): 243–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOIE: Questions and answers about influenza A (H7N9). World Organization for Animal Health 2013. Reference Source\n\nDi Giuseppe G, Abbate R, Albano L, et al.: A survey of knowledge, attitudes and practices towards avian influenza in an adult population of Italy. BMC Infect Dis. 2008; 8: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYupiana Y, de Vlas SJ, Adnan NM, et al.: Risk factors of poultry outbreaks and human cases of H5N1 avian influenza virus infection in West Java Province, Indonesia. Int J Infect Dis. 2010; 14(9): e800–e5. PubMed Abstract \| Publisher Full Text\n\nIndriani R, Samaan G, Gultom A, et al.: Environmental sampling for avian influenza virus A (H5N1) in live-bird markets, Indonesia. Emerg Infect Dis. 2010; 16(12): 1889–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKandun IN, Tresnaningsih E, Purba WH, et al.: Factors associated with case fatality of human H5N1 virus infections in Indonesia: a case series. Lancet. 2008; 372(9640): 744–9. PubMed Abstract \| Publisher Full Text\n\nSedyaningsih ER, Isfandari S, Setiawaty V, et al.: Epidemiology of cases of H5N1 virus infection in Indonesia, July 2005-June 2006. J Infect Dis. 2007; 196(4): 522–7. PubMed Abstract \| Publisher Full Text\n\nAditama TY, Samaan G, Kusriastuti R, et al.: Avian influenza H5N1 transmission in households, Indonesia. PLoS One. 2012; 7(1): e29971. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKandun IN, Wibisono H, Sedyaningsih ER, et al.: Three Indonesian clusters of H5N1 virus infection in 2005. N Engl J Med. 2006; 355(21): 2186–94. PubMed Abstract \| Publisher Full Text\n\nChan PK, Lee N, Zaman M, et al.: Determinants of antiviral effectiveness in influenza virus A subtype H5N1. J Infect Dis. 2012; 206(9): 1359–66. PubMed Abstract \| Publisher Full Text\n\nAdisasmito W, Chan PK, Lee N, et al.: Effectiveness of antiviral treatment in human influenza A(H5N1) infections: analysis of a Global Patient Registry. J Infect Dis. 2010; 202(8): 1154–60. PubMed Abstract \| Publisher Full Text\n\nAdisasmito W, Aisyah DN, Aditama TY, et al.: Human influenza A H5N1 in Indonesia: health care service-associated delays in treatment initiation. BMC Public Health. 2013; 13(1): 571. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOjulong J, Mitonga KH, Iipinge SN: Knowledge and attitudes of infection prevention and control among health sciences students at University of Namibia. Afr Health Sci. 2013; 13(4): 1071–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVivas AP, Gelaye B, Aboset N, et al.: Knowledge, attitudes and practices (KAP) of hygiene among school children in Angolela, Ethiopia. J Prev Med Hyg. 2010; 51(2): 73–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSady H, Al-Mekhlafi HM, Atroosh WM, et al.: Knowledge, attitude, and practices towards schistosomiasis among rural population in yemen. Parasit Vectors. 2015; 8: 436. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbbate R, Di Giuseppe G, Marinelli P, et al.: Knowledge, attitudes, and practices of avian influenza, poultry workers, Italy. Emerg Infect Dis. 2006; 12(11): 1762–5. PubMed Abstract \| Free Full Text\n\nSarker S, Saranika T, Chowdhury EH, et al.: Isolation and Identification of Bacteria from the Workers of Live Bird Markets at Mymensingh, Bangladesh. J Biosci. 2009; 17: 135–8. Publisher Full Text\n\nOlsen SJ, Laosiritaworn Y, Pattanasin S, et al.: Poultry-handling practices during avian influenza outbreak, Thailand. Emerg Infect Dis. 2005; 11(10): 1601–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTong A, Sainsbury P, Craig J: Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int J Qual Health Care. 2007; 19(6): 349–57. PubMed Abstract \| Publisher Full Text\n\nAbbate R, Di Giuseppe G, Marinelli P, et al.: Knowledge, attitudes, and practices of avian influenza, poultry workers, Italy. BMC Infect Dis. 2006; 12(11): 1762.\n\nWang M, Han X, Fang H, et al.: Impact of health education on knowledge and behaviors toward infectious diseases among students in Gansu Province, China. Biomed Res Int. 2018; 2018: 6397340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBavel JJV, Baicker K, Boggio PS, et al.: Using social and behavioural science to support COVID-19 pandemic response. Nat Hum Behav. 2020; 4(5): 460–71. PubMed Abstract \| Publisher Full Text\n\nLau JT, Griffiths S, Choi KC, et al.: Widespread public misconception in the early phase of the H1N1 influenza epidemic. J Inf Secur. 2009; 59(2): 122–7. PubMed Abstract \| Publisher Full Text\n\nPark JH, Cheong HK, Son DY, et al.: Perceptions and behaviors related to hand hygiene for the prevention of H1N1 influenza transmission among Korean university students during the peak pandemic period. BMC Infect Dis. 2010; 10: 222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRubin GJ, Amlôt R, Page L, et al.: Public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey. BMJ. 2009; 339: b2651. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeung YY, Wong KY, Kwong WY, et al.: Severe acute respiratory syndrome outbreak promotes a strong sense of professional identity among nursing students. Nurse Educ Today. 2005; 25(2): 112–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcConnell I: One Health in the context of medical and veterinary education. Rev Sci Tech. 2014; 33(2): 651–7. PubMed Abstract \| Publisher Full Text\n\nPeng Y, Pei C, Zheng Y, et al.: A cross-sectional survey of knowledge, attitude and practice associated with COVID-19 among undergraduate students in China. BMC Public Health. 2020; 20(1): 1292. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRehman S, Rantam FA, Batool K, et al.: Knowledge, attitude and practices associated with Avian influenza among undergraduate university students of East Java Indonesia: A cross-sectional survey. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.16664488.v1" }	[ { "id": "121510", "date": "21 Feb 2022", "name": "Sohail Ahmad", "expertise": [ "Reviewer Expertise Poultry 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\nFirst of all, thank you very much for giving me the opportunity to review this article, I have completed my evaluation and after reading this article carefully I am of the opinion that this manuscript should be considered for indexing.\n\nThe idea is very well-conceived and presented clearly, objectives are well organized and results are completely justifying the hypothesis. In general, this manuscript provides sound information and a good contribution in the field of Poultry Science (Poultry Diseases). I have provided some further comments and queries here.\nThank you very much and please let me know if I can be of further assistance\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "123902", "date": "14 Mar 2022", "name": "Farazi Muhammad Yasir Hasib", "expertise": [ "Reviewer Expertise Veterinary Epidemiology", "Food Microbiology", "Food Safety", "One 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\nGeneral comments: The article was a questionnaire-based cross-sectional survey among undergraduate students (knowledge, attitude, and practices) in the mentioned study area on avian influenza. The report made it clear that the veterinary students learned more about this zoonotic disease than students of other faculties. The article preparation and structure are good, and they can be indexed. The manuscript has a clear layout, and the research findings fit the study objectives. Even though the author used clear English, there is enough scope to improvise sentence structures. Minor editing on references, tables, and English could improve the article.\nAbstract: The abstract seems highly well. Methods, results, and conclusions are correlated to the study and understandable.\nIntroduction:\nLiterature was clearly stated for publication, but some recent references would be good. A questionnaire-based survey and its importance should be a vital part of the introduction.\nMethods: Methods are comprehensive, and I do believe they cover the idea. The use of “Few” in the methodology is a little improper. How many students from the other faculty already knew about the disease would correlate with their response to the questionnaire.\nResults: The results could cover the objectives with the descriptive study, which could've done the regression analysis, but I think this could be enough for this report. However, the writing needs to be revised for more concise and precise. I would suggest one thing to consider - the author can make one pictorial presentation of the constructed tables.\nDiscussion: Excellently written already, add some new references, particularly previous 2/3 years. This part needs to be revised mainly keeping in mind that results should not be repeated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-115
https://f1000research.com/articles/10-192/v2	18 Mar 21	{ "type": "Research Article", "title": "Tailored PRISMA 2020 flow diagrams for living systematic reviews: a methodological survey and a proposal", "authors": [ "Lara A. Kahale", "Rayane Elkhoury", "Ibrahim El Mikati", "Hector Pardo-Hernandez", "Assem M. Khamis", "Holger J. Schünemann", "Neal R. Haddaway", "Elie A. Akl", "Lara A. Kahale", "Rayane Elkhoury", "Ibrahim El Mikati", "Hector Pardo-Hernandez", "Assem M. Khamis", "Holger J. Schünemann", "Neal R. Haddaway" ], "abstract": "Background: While the PRISMA flow diagram is widely used for reporting standard systematic reviews (SRs), it was not designed for capturing the results of continual searches for studies in living systematic reviews (LSRs). The objectives of this study are (1) to assess how published LSRs report on the flow of studies through the different phases of the review for the different updates; (2) to propose an approach to reporting on that flow. Methods: For objective 1, we identified all LSRs published up to July 2020. We abstracted information regarding their general characteristics and how they reported on search results. For objective 2, we based our proposal for tailored PRISMA approaches on the findings from objective 1, as well as on our experience with conducting Cochrane LSRs. Results: We identified 108 living publications relating to 32 LSRs. Of the 108 publications, 7% were protocols, 24% were base versions (i.e., the first version), 62% were partial updates (i.e., does not include all typical sections of an SR), and 7% were full updates (i.e., includes all typical sections of an SR). We identified six ways to reporting the study flow: base separately, each update separately (38%); numbers not reported (32%); latest update separately, all previous versions combined (20%); base separately, all updates combined (7%); latest update version only (3%); all versions combined (0%). We propose recording in detail the results of the searches to keep track of all identified records. For structuring the flow diagram, we propose using one of four approaches. Conclusion: We identified six ways for reporting the study flow through the different phases of the review for the different update versions. We propose to document in detail the study flow for the different search updates and select one of our four tailored PRISMA diagram approaches to present that study flow.", "keywords": [ "PRISMA statement", "living systematic review", "update", "research methodology research reporting", "flow chart", "systematic review reporting standards", "evidence synthesis", "research transparency", "research replication" ], "content": "Introduction\n\nDuring the coronavirus disease 2019 (COVID-19) pandemic, health research has proliferated exponentially1. Systematic reviews are essential to synthesize the evidence and inform policy and practice. Given the pace of research publication, those reviews need to be kept up to date. Living systematic reviews (LSRs) are an emerging type of systematic review that involves the continual search of the literature and incorporation of relevant new evidence, soon after it becomes available2. While many evidence synthesis groups are engaged in conducting LSRs or living network meta-analyses, others have developed living databases or living maps, including resources specific for COVID-19 literature3–17.\n\nAn essential component of systematic reviews is to keep track of and report the number of records captured while searching the scientific literature and details of the selection process18. The PRISMA statement recommends the use of the PRISMA flow diagram to depict the flow of studies through the different phases of the systematic review19. While the PRISMA flow diagram is a widely used tool for reporting original systematic reviews, it was not designed to capture the results of continual searches typically used in LSRs. Hence, it’s unclear how authors of LSRs address the issue of presenting results of these continual searches.\n\nThe objectives of this study were (1) to assess how published LSRs report on the flow of studies through the different phases of the review for the different updates; and (2) to propose an approach to documenting and reporting on the flow of studies through the different phases of a LSR, for the different updates.\n\n\nMethods\n\nFor objective 1, we collected relevant data as part of a larger methodological survey aiming to assess the methods of conduct and reporting of LSRs. We have described the details of that study in a previously published protocol20. Briefly, we identified all living reviews published up to July 2020 available from the following electronic databases: Medline, EMBASE and the Cochrane library (see extended data21 of Khamis et al.20 for the search strategy). An eligible living review was either (1) a protocol for an LSR, (2) a base version of an LSR, (3) a full update version of an LSR, (4) a partial update version of an LSR, or (5) a combination of any of these (e.g., one living review may constitute of a protocol, a base version, and a full update version; another living review may constitute of only a Box 1 the definition of each type of living reviews.\n\n\n\n• LSR protocol: the protocol that describes the planned methods of the living review\n\n• Base version: the first version of the review that follows a living approach\n\n• Full update version: a subsequent version of the review that includes all the typical sections of a systematic review, including an introduction, methods, and results sections. Such a version could stand-alone in terms of content.\n\n• Partial update version: a subsequent version of the review that does not include all the typical sections of a systematic review, but instead refers to a previous version for complementary information. Such a version could not stand-alone in terms of content.\n\nFor the current study, we abstracted information about the following features of LSRs:\n\nGeneral characteristics:\n\nPublication type, i.e., protocol, base version, full update version, partial update version.\n\nWhether published in the Cochrane library or elsewhere.\n\nField (e.g., clinical, public health)\n\nWhether COVID-19 related or not\n\nWhether the base version of the living review conducted as a rapid review or not\n\nReporting on study flow\n\nMethod used to report on the study flow (including the search results and the results of the selection process):\n\n▪ Narrative format and/or flow diagram.\n\n▪ Whether the results of the base and update searches are reported separately or not.\n\nType of flow diagram, if applicable (e.g., PRISMA).\n\nFor objective 2, we base our proposal for tailored PRISMA 2020 flow diagram approaches on the findings from objective 1, on our experience conducting Cochrane LSRs, and our methodological work on designing and reporting living evidence. Since 2017, our group has been responsible for the first series of three Cochrane LSRs, all of which address anticoagulation in patients with cancer22–24. We conducted the base search in February 2016. Since then, we have been updating the search on a monthly basis. Through this experience, we have been able to apply and refine the guidance for conducting LSRs endorsed by the living evidence network group25. Specifically, we explored solutions for the reporting of the study flow that would address different scenarios. Our goal was not to be prescriptive and narrow, but rather to cover all possible resulting flows by reviewing the LSRs we identified based on objective 1. Two authors developed a draft of the tailored approaches to presenting the study flow, and then circulated to the author team for review and suggestions for improvement.\n\n\nData handling and analysis\n\nWe used REDCap to collect and manage the data abstraction process. All data were exported from REDCap and analyzed using Stata v. 1326,27.\n\n\nResults\n\nOur search identified a total of 108 living publications relating to 32 LSRs. Table 1 shows their general characteristics. Of the 108 living publications, 8% were protocols, 24% were base versions, 61% were partial updates, and 8% were full updates. The median number of living publications per LSR was 1 (Interquartile range 1–4). Of the 32 living reviews, 31% were published in the Cochrane library, 30% were related to COVID-19, and 15% had a base version published as a rapid review. The majority were related to clinical topics (78%).\n\nAbbreviations: LSR: living systematic review; IQR: interquartile range\n\nTable 2 shows the results for the reporting on the study flow. Most base versions and full updates used a flow diagram to report on the search results (96% and 100% respectively), whereas none of the partial updates presented a flow diagram.\n\na When a flow diagram is not reported, the authors reported on the search results in a narrative format.\n\nAmong the 74 publications related to LSRs that included at least one update (12 LSRs) (Figure 1):\n\n38% reported the search results for the base version and for each update version separately.\n\n32% did not report the search results at all (e.g., ‘new studies identified and integrated’ without specifying the number).\n\n20% reported the search results for the latest update version separately and for all previous versions combined (including the base).\n\n3% reported the search results for the latest update version only.\n\n7% reported on the search results for the base version separately and for all update versions combined.\n\n0% reported the search results for all the different versions combined.\n\nUsing the approach described in the methods section, we developed four approaches that allow authors to document and report the study flow for the different review update versions of an LSR.\n\n1. Documenting LSR study flow\n\nAuthors should record in detail the results of the searches to keep track of all identified records. We propose using a spreadsheet for one LSR at a time. The format we present consists of tabs for each of the respective search sources: bibliographic databases (e.g. MEDLINE, EMBASE, Cochrane databases); conference proceedings; ongoing studies as captured in clinicaltrials.gov and WHO International Clinical Trials Registry Platform (ICTRP); other tabs as needed, and a final ‘cumulative’ tab.\n\nWe show in Figure 2 a snapshot of the ‘cumulative’ tab of the spreadsheet that keeps track of all records. It shows the study flow for a hypothetical example for an LSR published first in January 2020 (i.e. base version) and updated on a monthly basis up to August 2020. Each row corresponds to a different update version. The columns present the following information for each update (columns B to E): the number of records received, deduplicated, included at title and abstract screening, and included at full-text screening (i.e., newly included reports). Additional columns (F to I) present the distribution of the newly included reports as relating to either: (1) new studies, (2) previously included studies, (3) ongoing (unpublished) studies, or (4) preprints.\n\nAfter manually entering the information in the first five tabs (corresponding to the different search sources) the total is automatically computed in the ‘cumulative’ tab.\n\n2. Reporting LSR study flow\n\nThe proposed spreadsheet above can act as a basis for a tailored PRISMA flow diagram for LSRs. For structuring the flow diagram for LSR, one can select one out of four tailored PRISMA 2020 flow diagram approaches:\n\nApproach 1: presenting the search results of the different versions separately (i.e., base and each update separately) (Figure 3).\n\nApproach 2: presenting the search results for the different versions combined (i.e., including base and all update versions) (Figure 4).\n\nApproach 3: presenting the search results for the base version separately, and the results of all update version combined (Figure 5).\n\nApproach 4: presenting the results of the latest update version separately, and the results of all previous versions (including the base) combined (Figure 6).\n\nIn our Cochrane reviews, we applied the second proposal where we present the results for the different searches combined.\n\n\nDiscussion\n\nThis study found that authors of LSRs are not consistent in reporting on the flow of studies through the different phases of the review for the different update versions. Thus, we propose to document in detail the study flow for the different search updates and select one of four tailored PRISMA 2020 flow diagram approaches to present that study flow.\n\nTo our knowledge, this is the first methodological survey that assesses how LSR authors report on the flow of studies through the different phases of the review for the different update versions of LSRs. In addition, the research expertise on our team covers both living approach and regular updating of traditional SR. We believe that our assessment forms a vital baseline and allows us to propose best practices for visualization options to improve consistency whilst the production of LSRs is still at a relatively early stage. Indeed, this survey is part of a larger methodological survey aiming to assess the methods of conduct and reporting of LSRs20, that would allow us to update our findings in the future.\n\nAuthors tend more towards producing partial updates of LSRs rather than continually updating the full manuscript in a form of a full update. This might seem like a pragmatic approach particularly for a rapidly growing research field and when methods do not seem to change from one update to another. The heterogeneity observed in the ways LSR authors report on the study flow is likely to be explained by the lack of clear guidance on how to do so.\n\nWe built our proposal on the PRISMA 2020 flow diagram and provide four approaches to tailor the needs for continual searchers used in LSR. The fourth approach is the closest to the current PRISMA 2020 flow diagram as it presents the results of the latest update version separately and the results of all previous versions (including the base) combined.\n\nIn addition, we proposed three other different approaches to provide options to LSR authors and publishing journals. Whatever approach one decides to follow, for transparency purposes, the systematic reviewers should ideally archive previous versions of the flow diagram (e.g., in an appendix). One major challenge will be to accommodate a large number of updates in the same diagram; some approaches would work better than others in that case. Also, advanced information technology solutions may allow fitting a large number of updates, for example as shown in our web-based prototypes that allow ‘toggling’ between approaches. Once an approach is selected, one may develop an interactive flow diagram that have been designed to facilitate developing and communicating flow diagrams concisely28.\n\nAdvanced information technology can also be utilized to simplify updating and tracking the change in all LSR sections including the PRISMA diagram. It would be optimal to develop the base version in a certain platform where all SR and LSR sections are reported as units (i.e., title, authors, background, objectives, inclusion criteria, effect estimate for outcome x). With each update and for every unit, the author has the luxury to keep the same text (if no change has occurred) or edit (if change has occurred). Each unit can be updated in a differential speed based on certain criteria. The edits could be highlighted to visualize the change. For a certain section, one would easily have access to the entries in the previous versions and possibly visualize a trend across the different versions (i.e., cross-sectional view for that specific item). For example, dynamic documents can be developed using ‘R markdown’, a document preparation system, where static text can be combined with in-line code and ‘code chunks’ that produce instantly updatable documents given a modified input29.\n\nFuture research should pilot the proposed approaches for documenting the study flow and for structuring the living flow diagram. In addition, qualitative studies would be helpful to explore: (1) the feasibility and acceptability by LSR authors, publishers, and users towards the proposal; and, (2) what the end-users would like to see in an LSR update.\n\n\nConclusions\n\nLSR authors are not consistent in reporting the flow of studies through the different phases of the review for the different update versions. We propose to document in detail the study flow for the different search updates and select one of our four tailored PRISMA 2020 flow diagram approaches to present that study flow. Finally, improving the reporting of study flow in LSR methodology is essential for incorporating living evidence when developing living guidance, particularly in the context of an urgent response30,31.\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": "Acknowledgment\n\nWe would like to acknowledge Dr. Mathew Page for his revision for the manuscript.\n\n\nReferences\n\nNasrallah AA, Farran SH, Nasrallah ZA, et al.: A large number of COVID-19 interventional clinical trials were registered soon after the pandemic onset: a descriptive analysis. J Clin Epidemiol. 2020; 125: 170–178. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPage MJ, McKenzie M, Bossuyt P, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. 2020. Publisher Full Text\n\nKhamis AM, Kahale LA, Pardo-Hernandez H, et al.: Methods of conduct and reporting of living systematic reviews: a protocol for a living methodological survey [version 2; peer review: 2 approved]. F1000Res. 2019; 8: 221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhamis AM, Kahale LA, Pardo-Hernandez HJ, et al.: Search strategies. figshare. Journal contribution. 2019. Publisher Full Text\n\nAkl EA, Kahale LA, Hakoum MB, et al.: Parenteral anticoagulation in ambulatory patients with cancer. Cochrane Database Syst Rev. 2017; 9(9): CD006652. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKahale LA, Hakoum MB, Tsolakian IG, et al.: Anticoagulation for the long-term treatment of venous thromboembolism in people with cancer. Cochrane Database Syst Rev. 2018; 6(6): CD006650. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKahale LA, Hakoum MB, Tsolakian IG, et al.: Oral anticoagulation in people with cancer who have no therapeutic or prophylactic indication for anticoagulation. Cochrane Database Syst Rev. 2017; 12(12): CD006466. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrooker J, Synnot A, McDonald S, et al.: Guidance for the production and publication of Cochrane living systematic reviews: Cochrane reviews in living mode. Cochrane Collaboration. 2019. Reference Source\n\nStataCorp: Stata Statistical Software: Release 13. College Station, Editor. StataCorp LP: TX. 2013.\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; 42(2): 377–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaddaway N: SRflowdiagram: flow charts for systematic reviews and maps. 2020.\n\nHaddaway NR: DynamicSMapResults: template for designing updatable systematic map results text using R Markdown (Version 0.0.1). Zenodo. 2020. Publisher Full Text\n\nAkl EA, Morgan RL, Rooney AA, et al.: Developing trustworthy recommendations as part of an urgent response (1-2 weeks): a GRADE concept paper. J Clin Epidemiol. 2020; 129: 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchünemann HJ, Santesso N, Vist GE, et al.: Using GRADE in situations of emergencies and urgencies: certainty in evidence and recommendations matters during the COVID-19 pandemic, now more than ever and no matter what. J Clin Epidemiol. 2020; 127: 202–207. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "87481", "date": "28 Jun 2021", "name": "Lex Bouter", "expertise": [ "Reviewer Expertise Methodology", "epidemiology", "research integrity", "open science", "systematic review methods." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLiving Systematic Reviews (LSRs) are updated as new evidence becomes available and gained popularity during the Covid-19 pandemic. This manuscript describes the way PRISMA 2020 flow diagrams are handled in 32 LSRs with a view to recommend how this can best be done. The topic is relevant albeit a bit narrow and the manuscript is written clearly. However, there’re some issues that should to be solved in the next version of the manuscript.\nMajor issues\nIt’s disappointing that no clear recommendation but four alternative recommendations are given without any guidance which one to select when. That sounds a bit like ‘anything goes’. The reduction from the six approaches found in the LSRs to date to the four recommended is not very impressive. I was also surprised that the recommendations were solely based on the experience of the authors. Why is no attempt made to consult survey methodologists and end-users of LSRs, e.g. by performing a Delphi study? Also Cochrane Methods Groups and the editors of the Cochrane Handbook seem not to have been approached with a request to state their view on the issue.\n\nThe findings presented are part of a larger project on the methods of LSRs about which near to nothing is said in the manuscript. That makes one wonder whether this is not too small a part of the harvest to be optimally useful. Please explain why this element on flow charts is separated from the rest.\n\nThe data set is quite small: 32 LSRs of which 8 are only available as study protocol, 12 have only one (base) version, and 12 have one or more updates. Why is no indication provided of the corresponding imprecision, e.g. by presenting 95% confidence intervals?\n\nThe bottom half of table 2 presents how the study flow is reported among the 12 LSRs that got at least one update. I recommend to do this for all 32 LSRs included, assuming that when no update is yet reported the envisioned handling of flow charts should be specified in either the review protocol or the base version of the review.\n\nIs the work 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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7674", "date": "28 Jan 2022", "name": "Lara Kahale", "role": "Author Response", "response": "The Reviewers’ comments are in bold font and our replies in regular font. Extracts from the text are in italic fonts with changes underlined. We have indicated the sections where revisions have been made in our manuscript. Reviewer 1: Lex Bouter Living Systematic Reviews (LSRs) are updated as new evidence becomes available and gained popularity during the Covid-19 pandemic. This manuscript describes the way PRISMA 2020 flow diagrams are handled in 32 LSRs with a view to recommend how this can best be done. The topic is relevant albeit a bit narrow and the manuscript is written clearly. However, there’re some issues that should to be solved in the next version of the manuscript. Major issues 1. It’s disappointing that no clear recommendation but four alternative recommendations are given without any guidance which one to select when. That sounds a bit like ‘anything goes’. The reduction from the six approaches found in the LSRs to date to the four recommended is not very impressive. I was also surprised that the recommendations were solely based on the experience of the authors. Why is no attempt made to consult survey methodologists and end-users of LSRs, e.g. by performing a Delphi study? Also Cochrane Methods Groups and the editors of the Cochrane Handbook seem not to have been approached with a request to state their view on the issue. Thank you for your comment. This study is a methodological survey aiming to summarize what current LSR authors are reporting. Given that the full methodology of living systematic reviews is still emerging, we prefer not to make firm recommendations but lay out options. However, this study is part of a bigger project aiming to develop extension to the PRISMA 2020 statement for LSRs (please see registration form on EQUATOR network website: Equator Network. PRISMA for LSR – Extension of PRISMA 2020 for living systematic reviews. 2021; Accessed from https://www.equator-network.org/library/reporting-guidelines-under-development/reporting-guidelines-under-development-for-systematic-reviews/#LSR]) . Consistently with the Reviewer’s suggestions, we will be following the EQUATOR Network’s guidance for developing health research reporting guidelines which include, but is not limited to, engaging stakeholders, including methodologists, LSR end-users, Cochrane groups, journal editors, etc. in Delphi exercise. In addition, we will be doing a scoping review of current guidance documents and methods papers, and a qualitative study with stakeholders. We have elaborated about this research under implications for future research. 2. The findings presented are part of a larger project on the methods of LSRs about which near to nothing is said in the manuscript. That makes one wonder whether this is not too small a part of the harvest to be optimally useful. Please explain why this element on flow charts is separated from the rest. Thank you for your query. This paper is the first published study within the larger project which aims to explore how LSRs authors are currently reporting, conducting, and publishing LSRs. We cite the protocol of that project (Khamis 2019). This study focuses on reporting flow diagrams, another study will focus on general characteristics and delay in updating LSRs, and a third study will focus on methodological features of LSRs. We have now updated our results to include data from up to April 2021, and our dataset now includes 279 publications of 76 LSRs. 3. The data set is quite small: 32 LSRs of which 8 are only available as study protocol, 12 have only one (base) version, and 12 have one or more updates. Why is no indication provided of the corresponding imprecision, e.g. by presenting 95% confidence intervals? Thank you for your comment. Unfortunately, that was the state of the science. However, as noted in the previous comment, we have now updated our search to include results up to April 2021 Including data from 279 publications of 76 LSRs. We avoided including confidence intervals for the simplicity of presentation and given the small sample size. We are happy to include these if the editor prefers so. 4. The bottom half of table 2 presents how the study flow is reported among the 12 LSRs that got at least one update. I recommend to do this for all 32 LSRs included, assuming that when no update is yet reported the envisioned handling of flow charts should be specified in either the review protocol or the base version of the review. Thank you for your suggestion. We have checked and found the following (now added to the text): ‘None of the 279 living publications reported in their methods section how they plan to report on the study flow’." } ] }, { "id": "87133", "date": "22 Jul 2021", "name": "Sonia Hines", "expertise": [ "Reviewer Expertise Systematic reviews", "evidence-based 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\nThis is an interesting proposal to solve the problem of study flow reporting in living systematic reviews (LSRs). As LSRs increase in number, the methodology and reporting requirements need to be well described and usable.\nI am not sure these particular suggested approaches are the most practical. Spreadsheets are not easily inserted into publications, but it is a worthwhile question to be asking, and working from the existing publications is a good starting point.\nThe authors have done what they set out to do, but I suggest further work is needed before a recommended method of study flow reporting is settled upon.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7675", "date": "28 Jan 2022", "name": "Lara Kahale", "role": "Author Response", "response": "The Reviewers’ comments are in bold font and our replies in regular font. Extracts from the text are in italic fonts with changes underlined. We have indicated the sections where revisions have been made in our manuscript. Reviewer 2: Sonia Hines 1. This is an interesting proposal to solve the problem of study flow reporting in living systematic reviews (LSRs). As LSRs increase in number, the methodology and reporting requirements need to be well described and usable. I am not sure these particular suggested approaches are the most practical. Spreadsheets are not easily inserted into publications, but it is a worthwhile question to be asking, and working from the existing publications is a good starting point. Thank you for your positive feedback and practical advice. We agree that spreadsheets are not easily publishable, that is why we recommend using it as a tool for the LSR authors to document in detail the study flow as opposed to using it as a reporting tool. We now elaborate in the discussion how this spreadsheet would feed in an app (shinyapp) that creates nicely designed flows for these four approaches well suited for insertion into a publication. 2. The authors have done what they set out to do, but I suggest further work is needed before a recommended method of study flow reporting is settled upon. We agree with this comment. This study is a methodological survey aiming to summarize what LSR authors are currently reporting. As this is not a guidance document, we are not making any recommendations. However, this study is part of a bigger project aiming to develop extension to the PRISMA 2020 statement for LSRs. For that purpose, we will be following the EQUATOR Network’s guidance for developing health research reporting guidelines which include, but is not limited to, engaging stakeholders, including methodologists, LSR end-users, Cochrane groups, journal editors, etc. in a Delphi exercise. In addition, we will be doing a scoping review of current guidance documents and methods papers, and a qualitative study with stakeholders." } ] } ]	2	https://f1000research.com/articles/10-192
https://f1000research.com/articles/10-336/v1	04 May 21	{ "type": "Research Article", "title": "Brace versus cast following surgical treatment of distal radial fracture: a prospective randomised study comparing quality of recovery", "authors": [ "Irén Sellbrant", "Johanna Blomstrand", "Jon Karlsson", "Bengt Nellgård", "Jan Jakobsson", "Johanna Blomstrand", "Jon Karlsson", "Bengt Nellgård", "Jan Jakobsson" ], "abstract": "Background: Immobilisation following surgical treatment of distal radial fractures (DRF) is traditionally performed with a dorsal cast splint. There is an interest in changing the rigid cast to a removable brace. This can reduce the risk for cast-corrections, complications and improve recovery of function. The aim of the study was to compare quality of recovery (QoR) between brace and traditional cast for immobilisation during the first postoperative week. Methods: 60 patients with American Society of Anesthesiologists (ASA) physical status 1–3, scheduled for surgical treatment of DRF under a supraclavicular block (SCB) in a day-surgery setting were randomised into two groups of immobilisation post-surgery; brace (n=30) versus traditional cast (n=30). Study objectives were: differences in self-assessed QoR using the QoR-15 questionnaire, postoperative oral oxycodone consumption, perioperative time events and unplanned healthcare contacts one week postoperatively. Results: 54 patients, 46 females/eight males were included in the analysis; 27 with brace and 27 with traditional cast. QoR-15 sum median scores improved significantly from baseline/preoperative to day 7 (brace p=0.001, cast p=0.001) with no differences between the two groups. The only difference found was that patients in the brace group had significantly worse pain score 24-hours post-surgery (p=0.022). No significant differences were seen in sum median oxycodone consumption the first three postoperative days. No differences were found in perioperative events or unplanned healthcare contacts. Conclusions: Brace appears to be a feasible option to traditional cast for immobilisation following surgical treatment of DRF. The early QoR was similar in both groups apart from more pain in the brace group the first 24 postoperative hours.", "keywords": [ "brace", "cast", "distal radial fracture", "immobilisation after surgery", "postoperative oxycodone consumption", "quality of recovery", "QoR-15", "removable splint." ], "content": "Introduction\n\nDistal radial fracture (DRF) is one of the most common fractures.1 Fracture reduction and cast treatment is the common practice; however, if good fracture positioning cannot be achieved, open surgical correction and fixation is the most preferred option. Surgical treatment of DRF is commonly performed under either regional anaesthesia (RA) like a supraclavicular block (SCB) or general anaesthesia (GA). The currently preferred surgical treatment is volar-plate fixation followed by external cast application.2 The procedure is usually associated with moderate postoperative pain, although occasionally severe. Poor postoperative pain control may interfere with rehabilitation, delay recovery and adversely affect outcomes. Achieving early pain control may help to improve patient satisfaction and functional outcomes. The cast is usually changed to a removable and adjustable brace after two weeks. Rehabilitation support from an occupational therapist is valuable in order to achieve the best final functional outcome. The use of a brace for stabilisation following DRF has now gained increased interest, and studies have shown high patient acceptance.3-5 There is, however, sparse information on the effect of the use of brace following surgical treatment of DRF in adult patients.6,7\n\nSelf-assessed quality of recovery (QoR) questionnaires evaluating the postoperative course in a multi-dimensional perspective have gained increasing interest. To capture the QoR from the patients’ perspective, a variety of QoR instruments have been developed.8-14 One of these rating scales, the 40-item QoR-40, has been most extensively validated and demonstrates excellent psychometric properties. It has been translated and validated in different languages. Quality of recovery-15 (QoR-15) was developed to assess the recovery in a more simplified and user-friendly manner without reducing the quality of the instrument. It is a unidimensional measurement of QoR assessing five domains: pain, physical well-being, physical independence, psychological support and emotional state.15 Assessing perioperative interventions by means of QoR could provide a broader evaluation than merely recording the pain and analgesia consumption. Chazapis et al showed that QoR-15 is an acceptable and feasible outcome measure for day-surgery patients.16 Lyckner et al translated and culturally adapted the QoR-15 into Swedish and scores demonstrated acceptable validity, reliability and responsiveness; it can be performed in 3 minutes.17\n\nThe aim of the present study was to assess QoR and opioid consumption, following surgical treatment of distal radial fractures (DRF) comparing postoperative immobilisation with brace versus traditional cast, during the first postoperative week.\n\nWe hypothesised that a brace, applied directly after surgery, will give the same or better QoR as a traditional cast during the first postoperative week after surgical treatment of DRF in day surgery.\n\n\nMethods\n\nThis randomised study is a part of a randomised clinical trial approved by the Gothenburg Ethical Committee (May 31 2018; registration no 214-18). It was also registered in the Sahlgrenska University Hospital GDPR (General Data Protection Regulation) database on August 28, 2018. The study was conducted in accordance with the tenets of the 1964 Declaration of Helsinki. It was retrospectively registered in clinicaltrials.gov (NCT03749174) on November 21, 2018 with explicit information about start of patient inclusion: September 3, 2018. All patients aged between 18-78 years, with American Society of Anesthesiologists (ASA) physical status 1–3 and scheduled for day surgery of a DRF between September 3, 2018, and June15, 2020, at the Department of Anaesthesia and Intensive Care, Sahlgrenska University Hospital/Mölndal Hospital, Gothenburg, Sweden, were assessed for eligibility. Written informed consent with permission to publish was obtained from all patients before enrollment.\n\nOpioid-naïve patients were included; when having a closed DRF assessed on radiographs and classified as AO 23 A-C1 (Orthopaedic Trauma Association), ≤17 days from trauma and scheduled for operative fixation with a locked volar-plate. Finally, maximum length-of-surgery had to be <90 min and all surgeons used tourniquet. Exclusion criteria were: multifractures, inflammatory diseases, dementia, severe psychiatric disorder or other cognitive dysfunction, ongoing drug and alcohol abuse, known local anaesthetic allergy, pregnancy and finally, no fluency of the Swedish language.\n\nFracture classification was performed by an experienced orthopaedic surgeon.\n\nIn total, 142 patients were informed about the study. 22 patients declined to participate, leaving 120 patient to be included following written informed consent. The effect of anaesthetic technique was assessed in 90 patients and is presented separately elsewhere. 60 patients with the same anaesthetic technique (SCB with mepivacaine), were randomised by the investigater, using sequentially numbered opaque envelopes, to one of two groups of immobilisation methods post-surgery: 1) traditional dorsal cast (n = 30); and 2) removeable brace (n = 30), prefabricated and stabled with volar and dorsal steel rails (Wrist lacer, Camp Scandinavia AB).\n\nAll patients followed the dedicated day-care bundle for enhanced recovery and safe discharge on the day of surgery. Perioperative care was optimised for providing rapid and effective recovery.\n\nThe QoR was measured with the QoR-15 score, (see the extended data).17 The questionnaire uses an 11-point numerical rating scale (for positive items, 0 = “none of the time” to 10 = “all of the time”; for negative items the scoring is reversed; maximum score 150). The 11-point numerical rating scale leads to a minimum score of 0 (very poor recovery) and a maximum score of 150 (excellent recovery). Question 7: “Getting support from hospital” was not useful in this study since all patients were in a day-surgery setting. That reduced the maximum total sum score to 140 points instead of 150. The five dimensions of health were incorporated in the 14 questions (questions 1–6 and 8–15): physical comfort (questions 1–4 and 13), physical independence (questions 5 and 8), psychological support (question 6), pain (questions 11 and 12) and emotions (questions 9, 10, 14, and 15).\n\nAll patients received oral premedication, acetaminophen (1000 mg), oxycodone (5 or 10 mg; 5 mg to >70 years and/or <60 kg), etoricoxib (90 mg; if no contraindication) and meclizine (25 mg). All patients were given 8 mg betamethasone intravenous perioperatively.\n\nAll patients underwent surgical treatment with a volar-plate fixation by a senior orthopaedic surgeon and were then immobilised for two weeks with one of the two randomised immobilisation methods applied directly post-surgery. After two weeks, the patients allocated to the traditional cast had this routinely replaced with a brace. All patients obtained a SCB with a short-acting local anaesthetic agent (mepivacaine 1%, 25-30 mL) and were offered a mild sedation with propofol perioperatively. SCB was performed by an experienced anaesthesiologist with ultrasound guidance.\n\nPatients being considered stable and adequatly pain-relieved bypassed the Post-Anaesthesia Care Unit (PACU) and were transferred directly to the post-surgery ward. Patients needing monitoring stayed in the PACU until considered stable. All patients were planned to be discharged home from the step-down ward.\n\nPatients obtained a protocol to note the type, dose and frequency of analgesic consumption at home and they all received the same postoperative pain management at discharge: oxycodone 5–10 mg and acetaminophen 1000 mg for pain control, respectively. They received a prescription of these medications to be taken ad libitum within a daily maximum dose of 30–40 mg oxycodone and 4000 mg of acetaminophen.\n\nAt day 2–4 postoperatively, the patients had their first appointment to the occupational therapist with following appointments scheduled at 2, 6, 12 weeks and 1 year postoperatively.\n\n\nData collection\n\nAll data were collected by the same two investigators, while patients were still in hospital and then by four follow-up telephone calls at 24, 48, 72 hours and 7 days after discharge. Patient characteristics were collected prior to start of surgery and anaesthesia. Pain assessment (NRS) and QoR-15 were performed four times (baseline/preoperatively, 24, 72 hours and 7 days post-surgery). Postoperatively, we collected oxycodone consumption, administered at the hospital and after discharge; the first 3 postoperative days and 7 days after surgery. We also registered perioperative time events; surgery time (including fixation with cast or brace), time the anaesthetic nurse was occupied with the patient, theatre time, unplanned admission, number of patients needing PACU stay, total time in hospital, SCB total duration time and duration time after surgery.\n\nThe primary outcome was difference in sum median (interquartile range) and its five domains of QoR-15 score at baseline, 24 hours, 72 hours and 7 days after surgery between the two groups.\n\nThe secondary outcome was sum median (interquartile range) of postoperative oxycodone consumption administered at the hospital and after discharge, the first 3 postoperative days and 7 days after surgery. Finally, perioperative time events were as described above.\n\nNumerical data are presented as mean and standard deviation (SD) and median and quartiles for non-normally distributed data. Categorical data is presented as numbers and percent. Differences between the the study groups, brace and cast, were studied with independent sample t-test for normally distributed data and Mann–Whitney U-test for skewed data. Differences in proportion were studied with a Chi-squared test. The QoR data are presented as median and interquartile range (IQR). Differences between sum median QoR-15 and changes in sum QoR-15 between time points were analysed with non-parametric tests, Mann–Whitney U-test and Kruskal–Wallis test as applicable. A p < 0.05 was considered statistically significant.\n\n\nResults\n\n60 patients scheduled for surgical treatment of DRF were included in the study following informed consent. Six patients were lost to follow-up; five due to failed supraclavicular block and one because of anatomic anomaly (Figure 1).\n\nFlow of patients through the trial. General anesthesia (GA).\n\n54 patients, 46 females and eight males with a mean age of 56 (SD ± 15) years, ASA 1–3 patients, were included in the analysis, 27 patients in each group; brace versus cast for immobilisation post-surgery. The mean age was lower (p = 0.02) and the mean BMI was higher (p = 0.02) in the brace group. No further differences were found in patients’ baseline characteristics (Table l).\n\nClassification of patients´ health and comorbidity level by the American Society of Anesthesiologists (ASA) system, Body mass index (BMI), Apfel score; riskfactors (1-4) for PostOperative Nausea and Vomiting (PONV).\n\nTime from injury and perioperative time events did not differ between the groups. The majority of patients could bypass the PACU and were transferred directly to the step-down ward. The SCB resolution-time was a mean of 2.6 hours after surgery in both groups. No futher differences were seen between the groups in any perioperative time events (Table 2).\n\nPost Anaesthesia Care Unit, (PACU), Day Surgery (DS), Supraclavicular block (SCB).\n\nTwo patients (7.4%) from the cast group were admitted overnight post-surgery, both due to social reasons. None of the patients in the brace group needed an unplanned overnight admission and neither did any patient need unplanned healthcare contacts after hospital discharge during the first postoperative week.\n\nSum median QoR-15 was equal in the two groups at preoperative baseline assessment. Then, there were an increase in sum median QoR-15 score in both study groups from baseline and up to 1 week after surgery (cast p = 0.001, brace p = 0.001). No postoperative reduction in sum median QoR-15 score was seen. There was a slightly different pattern between the two groups and thus, the cast group had the largest increase from baseline already after 24 hours while the brace group increased slower and reached the largest increase from baseline first at 72 hours post-surgery. The sum median QoR-15 score did not differ between the groups one week postoperatively (Figure 2, Table 3).\n\nMaximum score possible was 140 points. Both groups showed an increase in sum median QoR-15 score from baseline and up to 1-week post-surgery (cast p = 0.001, brace p = 0.001).\n\nThe preoperative/baseline QoR-15-sum-score showed no difference between the groups. However, the domain physical comfort was significantly lower (p = 0.048) in the cast group. The different domains in the QoR-15 questionnaire showed only minor differences between the two groups at the four time-points studied. There was a significant difference in pain-score at 24 hour-assessment when brace scored worse than cast (p = 0.022) (Figure 3, Table 3).\n\nMedian postoperative oxycodone consumption was low overall. Median consumption for both groups together was 27.7 (IQR 9-46) mg the first three postoperative days. No differences in oxycodone consumption were seen (Table 4).\n\n\nDiscussion\n\nThis study was initiated to assess the feasibility to use a removable brace compared with a traditional cast following surgical treatment of distal radial fracture (DRF). There are two main findings in the present study:\n\nFirstly, supported our hypothesis, we found no significant difference in the sum QoR-15 score, merely a minor difference was seen in subdomain pain, noted at 24 hours, between the brace and cast groups in QoR during the first postoperative week. The multifactorial reasons of the experienced pain have not been furthermore investigated in this study.\n\nSecondly, we found no deterioration in the sum median QoR-15 score post-surgery in neither of the two groups. The postoperative sum median QoR-15 score increased over time from baseline until one-week post-surgery in both study groups, with no differences at any time-point assessed. This is a scarce finding since most patients usually do show impairment at 24 hours post-surgery.16 This could indicate that the preoperative QoR-15 score may not be a true baseline score. Focusing on individual items of the scores, the results indicated that patients were tired, anxious and in pain 24 hours prior to surgery. Further, all the patients in the present study had their trauma in average 9 days prior to surgery and most of them had been treated conservatively without success. These circumstances may not provide an ideal baseline for assessing recovery following surgery and could explain the impaired QoR-15 baseline scores; however, it may still constitute a baseline for comparison. This can thus explain the result that we found no postoperative deterioration in the sum median QoR-15 score at 24 hours post-surgery, merely a continuous improvement. Chazapis et al. had similar result in their study assessing orthopaedic patients scheduled for day surgery (DS).16\n\nBoth study groups had a SCB with a short-acting local anaesthetic agent (mepivacaine) as part of a multi-modal analgesia concept, reducing the risk for rebound pain after discharge.18 Oral pain therapy was started before surgery, where all patients had acetaminophen, etoricoxib, oxycodone given preoperatively and 8 mg betamethasone given intravenously perioperatively as a part of the multi-modal analgesic treatment. Thus, the difference in early pain is possibly related to the brace. The numerically higher oxycodone consumption, though not statistically significant, may at least partly be associated to oxycodone dosage, adjusted to age and BMI, since patients with brace were both significantly younger and had higher BMI. No further differences were seen in the patients’ self-assessed QoR or oxycodone consumption.\n\nDay surgery (DS) continues to grow as a field of perioperative care and now also subacute surgeries, like fractures, are performed as day surgery. None of the patients in the present study required hospital admission. Two patients were however addmitted of social reasons, not related to recovery. There were no unplanned healthcare contacts during the first postoperative week and therefore, this study confirms the feasibility of scheduling surgical treatment of DRF as a day surgery procedure. The immobilisation techniques hardly impact day surgery planning, nor may it affect theatre times. Although we found no time gain in the brace-group, we speculate that using a brace should reduce time in theatre as we avoid the time for cast construction. Every intervention that could facilitate a rapid, safe and effective patient turnover is of importance.\n\nThe ability to resume normal activities of daily living after surgery and anaesthesia is an important indicator of a high quality of perioperative care. Most patients’ QoR-15 scores had returned to their preoperative values and exceeded them after 24 hours. This randomised study suggests that measurement of QoR-15 before surgery, (but not on the day of surgery), and 48 h postoperatively could provide a useful and feasible assessment of patient-reported outcome after day case orthopaedic surgery.16\n\nThis study had a prospective randomised clinical design that minimised the risk of confounding factors. It was a single-centre design without any loss to follow-up reducing the risk of selection and information bias and also warrants generalisability of this study. Only two investigators collected the data ensuring consistency and high standard of data collection.\n\nHowever, there are some limitations to this study. All results must be assessed remembering the fact that no sample size calculation was made for this particular study. The study is a part of a larger study assessing anaesthetic technqiues and the sample size calculation was made for that study.\n\nThe aim of QoR-15 was to assess QoR following anaesthesia and surgery and not to assess QoR following different immobilisation techniques. The sensitivity of the QoR-15 instrument may have been to low for this intervention and a potential ceiling effect must be acknowledged. Moreover, the trial was not blinded to any of the anaesthesia/surgery staff, nor to the study nurse and nor to the patient. We excluded patients with poor Swedish comprehension and severe pre-existing medical conditions. We can not disregard the fact, that a nurse repeatedly calling the patients to ask for their health status during the first postoperative week could contribute to a terapheutic effect.\n\n\nConclusion\n\nWe found brace, applied directly after surgery, to be a feasible option to traditional cast for immobilisation following surgical treatment of DRF. There was no difference in quality of recovery, assessed by QoR-15, during the first postoperative week, between brace and cast. The effects of brace immobilisation on more protracted recovery and wrist functions needs further studies.", "appendix": "Acknowledgements\n\nThe authors wish to thank study nurse A-C Ridderstråle, Dr A Bojan and Dr C Olsen for their invaluable help with data collection. Dr M Brattwall for stimulating the start of this work. All at Sahlgrenska University Hospital/Mölndals hospital.\n\n\nData availability\n\nOSF: Underlying data for ‘Brace versus cast following surgical treatment of distal radial fracture: a prospective randomised study comparing quality of recovery’, https://doi.org/10.17605/OSF.IO/KJC2N.19\n\nThis project contains the following underlying data: pain scores, oxycodone consumption and QoR15 scores\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).\n\nOSF: CONSORT checklist for ‘Brace versus cast following surgical treatment of distal radial fracture: a prospective randomised study comparing quality of recovery’, https://doi.org/10.17605/OSF.IO/KJC2N.19\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).\n\n\nReferences\n\nJerrhag D, Englund M, Karlsson MK, et al.: Epidemiology and time trends of distal forearm fractures in adults - a study of 11.2 million person-years in Sweden. BMC Musculoskelet Disord. 2017; 18(1): 240. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMellstrand-Navarro C, Pettersson HJ, Tornqvist H, et al.: The operative treatment of fractures of the distal radius is increasing: results from a nationwide Swedish study. Bone Joint J. 2014; 96-b(7): 963–9. PubMed Abstract \| Publisher Full Text\n\nMcVeigh KH, Berger TG, Cudahy R, et al.: An Evidence-Based Approach to Casting and Orthosis Management of the Pediatric, Adolescent, and Young Adult Population for Injuries of the Upper Extremity: A Review Article. Clin J Sport Med. 2019. PubMed Abstract \| Publisher Full Text\n\nCui Z, Yu B, Hu Y, et al.: Dynamic versus static external fixation for unstable distal radius fractures: an up-to-date meta-analysis. Injury. 2012; 43(7): 1006–13. PubMed Abstract \| Publisher Full Text\n\nFoster BD, Sivasundaram L, Heckmann N, et al.: Distal Radius Fractures Do Not Displace following Splint or Cast Removal in the Acute, Postreduction Period: A Prospective. Observational Study. J Wrist Surg. 2017; 6(1): 54–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStuby FM, Döbele S, Schäffer SD, et al.: Early functional postoperative therapy of distal radius fracture with a dynamic orthosis: results of a prospective randomized cross-over comparative study. PLoS One. 2015; 10(3): e0117720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHandoll HH, Huntley JS, Madhok R: Different methods of external fixation for treating distal radial fractures in adults. Cochrane Database Syst Rev. 2008 (1): Cd006522. PubMed Abstract \| Publisher Full Text\n\nGornall BF, Myles PS, Smith CL, et al.: Measurement of quality of recovery using the QoR-40: a quantitative systematic review. Br J Anaesth. 2013; 111(2): 161–9. PubMed Abstract \| Publisher Full Text\n\nStark PA, Myles PS, Burke JA: Development and psychometric evaluation of a postoperative quality of recovery score: the QoR-15. Anesthesiology. 2013; 118(6): 1332–40. PubMed Abstract \| Publisher Full Text\n\nMyles PS, Hunt JO, Nightingale CE, et al.: Development and psychometric testing of a quality of recovery score after general anesthesia and surgery in adults. Anesth Analg. 1999; 88(1): 83–90. PubMed Abstract \| Publisher Full Text\n\nRoyse CF, Newman S, Chung F, et al.: Development and feasibility of a scale to assess postoperative recovery: the post-operative quality recovery scale. Anesthesiology. 2010; 113(4): 892–905. PubMed Abstract \| Publisher Full Text\n\nRoyse CF, Williams Z, Purser S, et al.: Recovery after nasal surgery vs. tonsillectomy: discriminant validation of the Postoperative Quality of Recovery Scale. Acta Anaesthesiol Scand. 2014; 58(3): 345–51. PubMed Abstract \| Publisher Full Text\n\nRoyse CF, Williams Z, Ye G, et al.: Knee surgery recovery: Post-operative Quality of Recovery Scale comparison of age and complexity of surgery. Acta Anaesthesiol Scand. 2014; 58(6): 660–7. PubMed Abstract \| Publisher Full Text\n\nLindqvist M, Granstrom A, Schening A, et al.: Cognitive testing with the Post-Operative Quality of Recovery Scale in pre-surgery cancer patients--a controlled study. Acta Anaesthesiol Scand. 2015; 59(6): 763–72. PubMed Abstract \| Publisher Full Text\n\nKleif J, Waage J, Christensen KB, et al.: Systematic review of the QoR-15 score, a patient- reported outcome measure measuring quality of recovery after surgery and anaesthesia. Br J Anaesth. 2018; 120(1): 28–36. PubMed Abstract \| Publisher Full Text\n\nChazapis M, Walker EM, Rooms MA, et al.: Measuring quality of recovery-15 after day case surgery. Br J Anaesth. 2016; 116(2): 241–8. PubMed Abstract \| Publisher Full Text\n\nLyckner S, Boregard IL, Zetterlund EL, et al.: Validation of the Swedish version of Quality of Recovery score-15: a multicentre, cohort study. Acta Anaesthesiol Scand. 2018; 62(7): 893–902. PubMed Abstract \| Publisher Full Text\n\nLavand'homme P: Rebound pain after regional anesthesia in the ambulatory patient. Curr Opin Anaesthesiol. 2018. PubMed Abstract \| Publisher Full Text\n\nJakobsson J: Brace vs cast.2021, April 14. Publisher Full Text" }	[ { "id": "86216", "date": "03 Jun 2021", "name": "Jakob Walldén", "expertise": [ "Reviewer Expertise Anesthesiology", "postop-recovery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 and structured manuscript evaluating postoperative quality of recovery (QoR) after surgical treatment of distal radius fracture under peripheral blockade. The authors compare immobilization with dorsal splint cast vs. removable brace and as main outcome measures uses self accessed QoR, opioid consumption. The study is an analysis of a subgroup in the RADAR-study (NCT03749174) and the randomization procedure is clearly pre-defined. Underlying data is accessible. The reporting follows CONSORT and statistical methods are appropriate. They find no main differences and concluded that brace is a feasible option. Conclusions are clear and justified.\nHowever, I have some concerns with the study that needs to be addressed:\n1. The study is a sub-analysis of two out of four arms in the RADAR-study, according to the registration in clinical trials. Please expand the description of the main study in the methods section and justify the decision for this sub-analysis as a separate manuscript. Further, how was the quality of recovery in the two other intervention arms? Please comment.\n2 The main outcome measure (QoR-15) is not mentioned in the clinical trial registration. Why? Please explain and also confirm that you had ethical approval to use the QoR-15 as your evaluation tool and that the approval was dated before the inclusion of patients in this prospective study.\n3. Pain assessment with NRS is one of two primary outcome variables in the predefined protocol according to www.clinicaltrials.gov. It is described in the methods section, but results not reported in the manuscript. Please explain/revise.\n\n4. Further, why was the occupational therapist evaluation on the 3rd day not included in the analysis? Please explain/discuss.\n5. The use of \"sum\" in the variables might be confusing for the readers of the paper. The QoR-15 score is the sum of all domains and you do not need to add \"sum\". Please revise all section in the manuscript. For the variable opioid consumption, it is better to use \"Total\" instead of \"sum\" (ex: Table 4).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7457", "date": "28 Jan 2022", "name": "Irén Sellbrant", "role": "Author Response", "response": "Reviewer Comments: Reviewer 1 (Jakob Walldén) It is a well written and structured manuscript evaluating postoperative quality of recovery (QoR) after surgical treatment of distal radius fracture under peripheral blockade. The authors compare immobilization with dorsal splint cast vs. removable brace and as main outcome measures uses self-accessed QoR, opioid consumption. The study is an analysis of a subgroup in the RADAR-study (NCT03749174) and the randomization procedure is clearly pre-defined. Underlying data is accessible. The reporting follows CONSORT and statistical methods are appropriate. They find no main differences and concluded that brace is a feasible option. Conclusions are clear and justified. However, I have some concerns with the study that needs to be addressed: 1. The study is a sub-analysis of two out of four arms in the RADAR-study, according to the registration in clinical trials. Please expand the description of the main study in the methods section and justify the decision for this sub-analysis as a separate manuscript. Authors Comments: The main RADAR-study was performed to assess different anaesthetic techniques and their impact on postoperative pain (NRS) and opioid consumption during the first 3 postoperative days. The RADAR-study arms assessing the impact of immobilization techniques was aimed to compare traditional cast with a flexible brace already as early fixation, directly post-surgery. Our interest was to assess whether the brace could improve the day case perioperative course, speeding up the perioperative time events and reducing early postoperative discomfort commonly associated to the cast fixation. The occupational therapist, also PhD-student, had the goal to assess the effects on the rehabilitation, need for unplanned visits due to cast issues and the functional outcome for up to 12-month post-surgery. Further, how was the quality of recovery in the two other intervention arms? Please comment. Authors Comments: The main object for the “main RADAR-study” was pain and opioid requirement, assessing the risk for rebound pain following block resolution and subsequent increased need for opioid analgesic during the intermediate postoperative course. The study aims were impact of the pain 24 hours post-surgery and during the first 3 postoperative days and opioid requirement during the same period, PONV/PDNV, time events, time to discharge and unplanned health contacts. We did unfortunately not focus on the quality of recovery in that part of the study. 2. The main outcome measure (QoR-15) is not mentioned in the clinical trial registration. Why? Please explain and also confirm that you had ethical approval to use the QoR-15 as your evaluation tool and that the approval was dated before the inclusion of patients in this prospective study. Authors Comments: The ethical review protocol was approved May 31st 2018 (as described in the method section) and contains QoR-15, The Clinical trial Registration was unfortunately not fully aligned with the ethical application, but is now updated accordingly clinicaltrials.gov (NCT03749174). 3. Pain assessment with NRS is one of two primary outcome variables in the predefined protocol according to www.clinicaltrials.gov. It is described in the methods section, but results not reported in the manuscript. Please explain/revise. Authors Comments: The primary of this part of the study was the QoR-15 questionnaire, time events during the perioperative course and the opioid consumption. We assessed pain in this subgroup by the QoR questions that showed a pain difference at 24 hours in favor for the traditional cast. 4. Further, why was the occupational therapist evaluation on the 3rd day not included in the analysis? Please explain/discuss. Authors Comments: The occupational therapist follow-up is still not completed (partly because of the COVID-19 Pandemic) and will commence up to 12 months’ post-surgery. The more in depth assessment of wrist function will hopefully, although delayed, be performed by the occupational therapists during coming autumn and winter. 5. The use of \"sum\" in the variables might be confusing for the readers of the paper. The QoR-15 score is the sum of all domains and you do not need to add \"sum\". Please revise all section in the manuscript. For the variable opioid consumption, it is better to use \"Total\" instead of \"sum\" (ex: Table 4). Authors Comments: Corrected accordingly." }, { "c_id": "7736", "date": "28 Jan 2022", "name": "Irén Sellbrant", "role": "Author Response", "response": "Reviewer 1 (Jakob Walldén) It is a well written and structured manuscript evaluating postoperative quality of recovery (QoR) after surgical treatment of distal radius fracture under peripheral blockade. The authors compare immobilization with dorsal splint cast vs. removable brace and as main outcome measures uses self-accessed QoR, opioid consumption. The study is an analysis of a subgroup in the RADAR-study (NCT03749174) and the randomization procedure is clearly pre-defined. Underlying data is accessible. The reporting follows CONSORT and statistical methods are appropriate. They find no main differences and concluded that brace is a feasible option. Conclusions are clear and justified. However, I have some concerns with the study that needs to be addressed: 1. The study is a sub-analysis of two out of four arms in the RADAR-study, according to the registration in clinical trials. Please expand the description of the main study in the methods section and justify the decision for this sub-analysis as a separate manuscript. Authors Comments: The main RADAR-study was performed to assess different anaesthetic techniques and their impact on postoperative pain (NRS) and opioid consumption during the first 3 postoperative days. The main RADAD-study is now further clarified in the method section and aligned to the Clinical Trial registration. The RADAR-study arms assessing the impact of immobilization techniques was aimed to compare traditional cast with a flexible brace already as early fixation, directly post-surgery. This is a separate intervention. Our hypothesis was that brace fixation direct after surgery could improve the day case perioperative course, speeding up the perioperative time events and reducing early postoperative discomfort commonly associated to the cast fixation and facilitate activities of daily living. The occupational therapist, also PhD-student, had the goal to assess the effects on the rehabilitation, need for unplanned visits due to cast issues and the functional outcome for up to 12-month post-surgery. Further, how was the quality of recovery in the two other intervention arms? Please comment. Authors Comments: The object for the “main RADAR-study” was pain and opioid requirement, assessing the risk for rebound pain following block resolution and subsequent increased need for opioid analgesic during the intermediate postoperative course. The study aims were impact of the pain 24-hours post-surgery and during the first 3 postoperative days and opioid requirement during the same period, PONV/PDNV, time events, time to discharge and unplanned health contacts. We did unfortunately not focus on the quality of recovery in that part of the study. 2. The main outcome measure (QoR-15) is not mentioned in the clinical trial registration. Why? Please explain and also confirm that you had ethical approval to use the QoR-15 as your evaluation tool and that the approval was dated before the inclusion of patients in this prospective study. Authors Comments: The ethical review protocol was approved May 31:st 2018 (as described in the method section) and contains QoR-15. The Clinical trials Registration was not fully aligned with the ethical application, but is now updated accordingly clinicaltrials.gov (NCT03749174). 3. Pain assessment with NRS is one of two primary outcome variables in the predefined protocol according to www.clinicaltrials.gov. It is described in the methods section, but results not reported in the manuscript. Please explain/revise. Authors Comments: The primary of this part of the study was the QoR-15 questionnaire, time events during the perioperative course and the opioid consumption. We assessed pain in this subgroup by the QoR questions that showed a pain difference at 24 hours in favor for the traditional cast. However, the numerical difference was less than what has been shown as a clinical relevant difference by Myles et al. (references). This is now clarified in the method as well as in the discussion section. 4. Further, why was the occupational therapist evaluation on the 3rd day not included in the analysis? Please explain/discuss. Authors Comments: The occupational therapist follow-up is still not completed (partly because of the Covid-Pandemic) and will commence up to 12 months’ post-surgery. The more in-depth assessment of wrist function will hopefully, although, delayed be performed by the occupational therapists during coming autumn and winter. 5. The use of \"sum\" in the variables might be confusing for the readers of the paper. The QoR-15 score is the sum of all domains and you do not need to add \"sum\". Please revise all section in the manuscript. For the variable opioid consumption, it is better to use \"Total\" instead of \"sum\" (ex: Table 4). Authors Comments: Corrected accordingly." } ] }, { "id": "118393", "date": "17 Jan 2022", "name": "Robert Gvozdenovic", "expertise": [ "Reviewer Expertise Hand Surgery." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear authors, thank you for this submission. Your paper is of high evidence level, well-designed, well-written and easy to read. Nevertheless, I have some comments and questions:\nDespite a good study-design, there might be a potential bias if any differences in fracture-complexity patterns between the groups existed. What if the cast group, for instance, had more complex fracture patterns? Please explain this in details, if only AO 23 A-C1 were included.\n\nDoes that possibly mean that the results of this study can only have clinical relevance for this particular distal radius fracture pattern? Your conclusion is definitely not in accordance to this assumption. If relevant, correct the conclusion accordingly.\n\nSo, the question is if the brace can also be safely used for more complex distal radius patterns? Add, eventually how many percent of this particular fracture pattern is generally present among all distal radius fracture needing surgery.\n\nWhy this study used only one PROM and not others where minimally important clinical difference (MICD) is existing in the literature (DASH)? If you used them, than the proper sample analysis before the study probably could be foretaken.\n\nThe use of sum median for both the QoR-scores and for opioid use is confusing and make the results not clear.\n\nThe flowchart needs to be corrected in accordance to the text where you claim that 5 patients did not show up on the follow-up.\n\nThere were not statistically significant difference for the postoperative pain in disfavor of brace. Could this be clinically relevant?\n\nMention the eventual cost difference between brace and the cast, if it exists. In some part of the world this question is not insignificant.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7737", "date": "28 Jan 2022", "name": "Irén Sellbrant", "role": "Author Response", "response": "Reviewer 2 (Robert Gvozdenovic) Dear authors, thank you for this submission. Your paper is of high evidence level, well-designed, well-written and easy to read. Nevertheless, I have some comments and questions: Despite a good study-design, there might be a potential bias if any differences in fracture-complexity patterns between the groups existed. What if the cast group, for instance, had more complex fracture patterns? Please explain this in details, if only AO 23 A-C1 were included. Authors Comments: Thank you for this constructive concern. We have made sure from the start that there was no inclusion bias. We have included AO 23, A, B and C1 fractures. These subgroups cover the common distal radius fractures. These fractures are common in this age group, and it is important to be able to make general conclusions. We decided from the very start to exclude C2 and C3 fractures, as they are different type(s) of fracture(s) and call for much more extensive and different treatment. We are therefore confident that there are no major differences in fracture-complexity patterns between the groups. We would also like to mention that this is a prospective randomized study and accordingly, the randomization would take care of minor differences and level out the risk of different cofounders in the different groups. In other words, we have included and randomized fractures of similar complexity levels. This is a prospective randomized study following the inclusion criteria described in the Methods section. Taken together, 6 patients were excluded from analysis, none of these were fracture/surgery related factors while 5 were excluded due to failed block and 1 due to an anatomic deviation. Does that possibly mean that the results of this study can only have clinical relevance for this particular distal radius fracture pattern? Your conclusion is definitely not in accordance to this assumption. If relevant, correct the conclusion accordingly. Authors Comments: There is only limited information about the use of brace as early immobilisation following open reposition and fixation of distal radius fracture in literature and this is one of the main reasons why this study was performed. As mentioned above, this study included AO 23 A, B and C1 fractures. These are the common distal radius fractures in this age group. There is a high volume, and these fractures are a relatively homogenous group of patients and thus seemingly adequate for inclusion to a randomised study. However, we agree that it is not possible to provide robust comments on the generalisation of the findings. Further studies are needed to confirm its safe use, especially in relation to more complex and complicated fractures (C 2 and C3) as mentioned in the conclusion. So, the question is if the brace can also be safely used for more complex distal radius patterns? Add, eventually how many percent of this particular fracture pattern is generally present among all distal radius fracture needing surgery. Authors Comments: Distal radius fracture (DRF) is one of the most common fractures, with an annual incidence of approximately 25000/year in Sweden. Data from the Swedish fracture register shows that approximately 20.6% of acute DRF undergo surgical fixation primarily and 5.5% secondary surgical fixation. We are aware of that need for open reposition and surgical fixation increases with fracture complexity. We included up to C1 fractures as described above. We do not have explicit national percentage of surgical need for the group studied, but it is without doubt the most common fractures. However, we managed to find statistics for DRF classified to AO 23 C1-3, registered at Sahlgrenska Universityhospital/Mölndals hospital from 2012-2021, see below. C2 and C3, the more complex fractures that not are a part of this study, seems to be 39% of all DRF that needs surgery. That leaves 61% to be the patients in our study. DRF class % of all DRF % needs surgery C1 12.1 41.8 C2 7.6 71.1 C3 5.5 84.8 Why this study used only one PROM and not others where minimally important clinical difference (MICD) is existing in the literature (DASH)? If you used them, then the proper sample analysis before the study probably could be foretaken. Authors Comments: The aim of the study was to assess the quality of recovery with the use of a multi-dimensional validated questionnaire constructed by the Australian group, where Paul Myles is the senior researcher. This quality of recovery questionnaire, which was used in the present study has previously been assessed as an effective scale for studying the recovery following surgery/anesthesia and it includes 5 main domains. And this was the main purpose of the study. It would of course have been of interest to also gain insight to patient reported outcomes, using PROM questionnaire, however, DASH is not optimally suited for evaluation of the early phase following surgery. “Myles et al. defined in a study 2016 updated in 2021, the size of the minimum clinically meaningful difference to the patient. They recommend the value to be 6.0 and the largest difference between the groups that we noted in this study was 4.0. It is, however, important to know that a significant difference in QoR-points doesn´t necessary have to be of clinical value.” The use of sum median for both the QoR-scores and for opioid use is confusing and make the results not clear. Authors Comments: The sum was used to describe that the total score for all 14 questions were analyzed/compared over the time-point studied and between groups. Likewise, the sum describes the total amount of opioid use for the 3 postoperative days studied. We have amended the text accordingly. Please see the revised manuscript. The flowchart needs to be corrected in accordance to the text where you claim that 5 patients did not show up on the follow-up. Authors Comments: Thank you for important comment; 6 patients were not included in the analysis and this is unfortunately described as lost for follow-up in the text. These 6 patients are excluded in the flow chart adequately and the description of exclusion is now corrected in the result section. The wording is, however, incorrect and is now amended accordingly. There were not statistically significant difference for the postoperative pain in disfavor of brace. Could this be clinically relevant? Authors Comments: There was a statistically significant difference in QoR pain domain scores at 24 hours postop. The numerical difference of at least 6 has by the score developers, however, shown a clinically relevant difference in domain scores. We found merely a numerical difference of 4. Moreover, we did not see any differences in need for opioid analgesics. This has been further clarified in the method as well as the discussion sections. Myles PS, Myles DB, Galagher W, Chew C, MacDonald N, Dennis A. Minimal Clinically Important Difference for Three Quality of Recovery Scales. Anesthesiology. 2016;125(1):39-45. Myles PS, Myles DB. An Updated Minimal Clinically Important Difference for the QoR-15 Scale. Anesthesiology. 2021;135(5):934-5. Mention the eventual cost difference between brace and the cast, if it exists. In some part of the world this question is not insignificant. Authors Comments: We agree that the impact of cost associated to a new intervention must be acknowledged. The direct cost associated to the brace is higher as compared to a cast. However, at our department we routinely apply a brace following the initial 2-week period with cast. The brace applied direct after surgery in this study can thus be used throughout the period. Thus, the net direct cost would not be increased, but possibly decreased. Moreover, the resources for a cast application, and its eventual corrections, will reduce the total cost. It should be acknowledged that all patients were called for a follow-up interview at 4 occasions, enabling patients to raise questions." } ] } ]	1	https://f1000research.com/articles/10-336
https://f1000research.com/articles/11-110/v1	28 Jan 22	{ "type": "Research Article", "title": "COVID-19 home isolation and food consumption patterns: Investigating the correlates of poor dietary diversity in Lebanon: a cross-sectional study", "authors": [ "Maha Hoteit", "Hussein Mortada", "Ayoub Al-Jawaldeh", "Carla Ibrahim", "Rania Mansour", "Ayoub Al-Jawaldeh", "Carla Ibrahim", "Rania Mansour" ], "abstract": "Background: The unfurling COVID-19 pandemic has uncovered the defenselessness of the Lebanese food system leading to serious implication in maintaining a healthy sustainable lifestyle. Aim: The main purpose of this study is to examine the impact of the COVID-19 pandemic on food consumption patterns and dietary diversity of the Lebanese people. Methods: The online survey, completed between April and June 2020, consisted of a cross-sectional study on 2282 Lebanese participants (mean age: 29.36±12.221, 80.9% women) that was part of a survey across 38 different countries conducted by De Backer, C. et al. A food frequency questionnaire was used to investigate the consumption patterns along with the calculation of the Food Consumption Score (FCS), a proxy indicator of dietary diversity. Data collected on cooking attitudes, shopping, and food stock identify the community mitigation measures. Results: Home isolation due to COVID-19 induced an increase in the consumption of legumes and pulses (3.2%, p-value=0.001) and whole wheat groups (2.8%, p-value=0.03). In contrast, a decrease of 5.4%, 6.9%, 5.8%, 5.1%, 3.1%, 3.4% and 2.8% was observed in the consumption of fruits (p-value=0), vegetables (p-value=0), processed meats, poultry, and fish (p-value=0), other dairy products (p-value=0), sweet snacks (p-value=0.001), sugared beverages (p-value=0), fats and oils (p-value=0.001), respectively. The FCS decreased by 4.6%. As food-related behaviors, most cooking attitudes, and practices (10 out of 13) showed an amelioration during the lockdown and the proportions of food stocked have been changing since the start of the pandemic seeing higher amounts of pasta, rice or other grains, flour, and legumes/pulses stocked. Conclusion: To conclude, the hostile home isolation strategy followed to prevent the COVID-19 spread in Lebanon, came at a high nutritional cost, driving poor dietary diversity.", "keywords": [ "COVID-19", "lockdown", "Lebanon", "food consumption pattern", "food security", "food consumption score", "mitigation measures" ], "content": "Introduction\n\nThe food deprivation and acute hunger faced by 25.9% of the global population and around 67 million people across all countries, make the public health importance of food consumption patterns and food security indisputable.1,2 In conflict-affected countries, disruption in agriculture and trade lead to the increase in the price of a simple plate of food that can cost more a day’s wages.2 Nowadays, almost two years after the first case was discovered, the SARS-CoV-2 (COVID-19) virus has affected more than 220 million people in 188 countries, more than 14 million people in the Eastern Mediterranean Region and caused more than four million deaths globally and around 260 million people died in the Eastern Mediterranean area.3 In Lebanon, from January until September 2021, there have been more than 700,000 confirmed cases of COVID-19 with around 8000 deaths, reported to the World Health Organization (WHO).4\n\nAs a consequence of the pandemic, the loss of livelihoods and remittances led to food supply disruptions and lack of income limiting the access of households to nutritious food, mainly at poorer and vulnerable population levels.1 Furthermore, around 1.5 billion people were not able to afford a nutrient-dense diet in which essential nutritional requirements are met.1,2,5 At the regional level, Eastern Mediterranean countries are faced with scarce and dwindling natural resources amidst high urbanization rates, population increases, wars, climate change, sociopolitical crises6 and recently, the COVID-19 pandemic.7 Lebanon, a small, densely populated country in the heart of a conflict-torn region, is experiencing a severe economic crisis in addition to the protracted Syrian refugee crisis. Along with the already-struggling economy, the unexpected COVID-19 pandemic has had disastrous sequels for all population groups in Lebanon, including food and medicine shortages, business closures, unemployment, and a significant drop in wages in a country where the national currency has lost more than 90% of its value.8 Therefore, more Lebanese families are being pushed further into poverty due to a lack of urgent economic reforms, with nearly three million people in Lebanon in need of financial and social assistance.8 The price of the basic food basket jumped by 340 percent, worsening food insecurity in Lebanon, with most Lebanese households fearful they may run out of food.8 This is clearly emphasizing the need for a situational analysis of the different dimensions of food consumption patterns and diet diversity in Lebanon.\n\nThe lack of studies concerning the changes in food consumption, mitigation measures, and food challenges had made this issue all the more important. Thus, the purpose of this study is to examine the impact of COVID-19 pandemic on the food consumption patterns and the diet diversity indicators mainly the food consumption score (FCS) of people residing in Lebanon. This report analyzed data from a study by De Backer, C. et al.9 but had a much more in-depth focus on data specific to Lebanon allowing for much more detailed and specific conclusions.\n\n\nMethods\n\nFull details of the methods used across all of the countries involved can be found in the original cross-sectional study described by De Backer, C. et al.9 Below the method used in Lebanon by the Lebanese team is outlined.\n\nThe online survey consisted of a cross-sectional study that was conducted in 38 different countries. Data related to inhabitants of Lebanon that have participated in this survey has been selected for the sake of analysis in this study. Questions of the survey were available in native Arabic language as well as other languages extending choices for the respondents. The survey was kept open between April 17th and June 25th and consisted of multiple blocks of information of which only a few variables are used and reported in this paper. Eligible participants included in this study were aged 18 years or older and of both genders residing in Lebanon during the COVID-19 crisis. Convenience sampling was used to recruit respondents and the survey was advertised via different social network platforms (Facebook, Instagram, Twitter, WhatsApp) as well as academic networks. The survey was managed and collected by a team in Lebanon including Maha Hoteit, Hussein Mortada, Rania Mansour and Elissa Naim (see acknowledgment). The questionnaire consisted of a validated online survey to collect information related to different topics including: sociodemographic and economic information, lockdown measures, cooking attitudes, shopping, food stock, and food frequency consumption in term of food portions per week (The question asked was: “how often did you eat the following [portions of] foods? Please indicate how often you consumed at least one portion of the following foods and drinks”).44 Regarding questions related to cooking attitudes, shopping and food frequency consumption, respondents were asked to answer each question two times, indicating their behavior in both periods (before the COVID-19 pandemic and during the COVID-19 lockdown). FCS, which is a proxy indicator used to assess dietary diversity, was calculated using the frequency of consumption of different food groups by a household during the seven days before the survey. The calculation formula of the FCS is: (starches × 2) + (pulses × 3) + vegetables + fruit + (meat × 4) + (dairy products × 4) + (fats × 0.5) + (sugar × 0.5).18 The FCS was calculated for each of the respondents based on their answers to the food frequency questionnaire. Prior to calculating the FCS score, response options were merged forming the following two categories: Less or equal to 4 times per week and more or equal to 5 times per week. Two different FCS were calculated, the first one was based on the answers of respondents about food frequency consumption before the lockdown and the second one was based on their answers during the lockdown. Everyone was then classified as having a high FCS (if it is greater than 42) or low FCS (less than 42).18 To determine factors that may impact the FCS, a binary logistic regression was calculated. In this regression the FCS (high vs low) was the dependent variable. A backward approach was used and factors having a p-value<0.05 were considered significant. Odds ratio and its confidence interval were also calculated for each of the factors.\n\nRespondents’ characteristics classified as categorical variables were presented as frequencies (percentages) while means ± standard deviation (SD) were used for continuous variables. Different statistical tests were used: The chi-square test was used to investigate differences for categorical variables between groups (gender) while an independent t-test was applied for continuous variables. A p-value lower than 0.05 was considered significant. Statistical analysis was conducted on IBM SPSS Statistics (IBM Corp, SPSS statistics for Mac, Version 24. Armonk, New York) (RRID:SCR_019096).\n\nA consent form was attached at the beginning of the online survey and was required to be completed before continuing. The consent form protected participants by letting them know their rights and responsibilities while keeping their information confidential. Moreover, this study received the approval of the ethics committees at the University of Antwerpen (SHW_46) (the country leader of this project) and at all the concerned countries, including Lebanon (#CUER 22-2020) given that it was observational with respect of confidentiality and no traceability of respondents.\n\n\nResults\n\nThe survey was completed by a convenient sample of 2449 Lebanese participants. A total number of 167 participants were excluded from the study due to either: being aged below 18 years or not completing the survey. Thus, 2282 Lebanese participants were included in the present study.43 Among them, 80.9% were females. Most participants were either adults (25 to 46 years old, 52.1%) or youth (19 to 20 years old, 37.7%). A very low percentage of adolescents (18 years) (8.4%) and elderly people (1.8%) had registered (Table 1). The mean age of the respondents was 29.36 years with a SD of 12.221. Males who responded to this survey were significantly older than females who did (p-value=0) (Table 1). In regard to the educational level, around 40.4% of the respondents had a bachelor’s degree when analyzing both genders together. In addition, a significant higher percentage (41.1%) of females had a bachelor’s degree compared with males (37.4%) (p-value=0) (Table 1). The household composition was also analyzed before and during the lockdown in which most households were composed of three to five adults (more than 45%). This trend has been also observed in males and females each separately, except for female living in the same household before the lockdown in which the majority were composed of less than three female adults per household (47.1%) (Table 1). When looking at economic characteristics, a similar percentage of respondents were students (40.5%) and active workers (40.9%) while the minority were unemployed (18.6%) before the COVID-19 lockdown. The percentage of unemployed individuals had increased to 32.6% during the lockdown. This increase has been also observed for males (8.7% before lockdown to 28.5% during lockdown) and females (20.9% before lockdown to 33.5% during lockdown). Moreover, the COVID-19 lockdown has also induced a loss of income among 62.9% of respondents. This loss of income was significantly higher (p-value=0.007) for men (68.6%) compared to women (61.6%). In addition, most respondents, when taken either all together (73.8%) or categorized as males (74.3%) and females (73.7%), struggle to make their wages last a month. And most even struggle to earn enough money for shopping (65.6%, 63.1% and 66.1% for all respondents, men, and women respectively) (Table 1).\n\nTables 2 and 3 show the food groups consumption and the FCS in the overall population, in Lebanon.\n\n* Unprocessed meats: (refers to all mammalian muscle meat including beef, veal, pork, lamb, mutton, horse, and goat).\n\nFruits group\n\nRegarding the consumption of fruits (fresh or frozen), 57% of the Lebanese population consumed fruits equals or less than 4 times per week during the lockdown, compared to 51.6% before the lockdown (p-value=0). It was also observed that the percentage of people consuming fruits five times and more per week, was relatively higher before the lockdown (48.4%) compared to the consumption during the lockdown (43%; p-value=0) (Table 2).\n\nVegetables group\n\nAs seen in Table 2, it appears that 64% of the population had higher consumption of vegetables (more than five times per week) before the lockdown compared to 57.1% during the lockdown (p-value=0). This statistic lead to the increase in the number of people in the four times or less category observed during the lockdown (42.9% vs 36% before the lockdown; p-value=0).\n\nLegumes and pulses group\n\n81% of people living in Lebanon, from the current study, were consuming legumes and pulses equals or less than 4 times per week before the lockdown; this category saw a decrease during the lockdown (77.8%). Consequently, the percentage of people frequently consuming legumes and pulses (five times and more per week) increased during the pandemic by 3.2% (22.2% during vs 19% before; p-value=0.001) (Table 2).\n\nNuts group\n\nAs per Table 2, the percentage of people frequently consuming nuts and derivatives was higher before the lockdown (15.9% before vs 14.7% during), while, a slight decrease of 1.2% was observed during the lockdown; 85.3% of the population was consuming nuts and derivatives 4 times or less during the lockdown compared to before (84.1%). Yet, these results were not statistically significant (p-value=0.147).\n\nProcessed meat/poultry/fish/vegetarian alternatives\n\nThe frequency of consumption of processed meat/poultry/fish/vegetarian alternatives was higher (more than five times per week) before the pandemic (18.6% before vs 12.8% during; p-value=0). In addition, a decrease of 5.8% in the consumption of this food group (four times or less per week) was observed during the lockdown (87.2% during vs 81.4% before; p-value=0) (Table 2).\n\nUnprocessed fish, unprocessed poultry, and unprocessed meats\n\nIt was observed that the frequent consumption (five times and more per week) of unprocessed fish was higher during the pandemic (6.2% during vs 5.7% before) and less frequent consumption (equals to 4 times or less per week) was higher before the lockdown (94.3% before vs 93.8% during). Yet, the frequency of consumption of unprocessed poultry and meat remained unchanged before and during the lockdown (Table 2). However, these results were not statistically significant (fish-p-value=0.366, poultry-p-value=1 and meat-p-value=0.948).\n\nWhite and whole wheat, bread, pasta, and grains\n\nThe frequency of consumption of wholewheat bread, pasta and grains was higher during the lockdown (25.8% during vs 23% before), yet this consumption was lower before the lockdown; 77% of the Lebanese population was consuming this food group equals to four times or less before the lockdown compared to 74.2% during the lockdown (p-value=0.003). As for the frequency of consumption of white bread, pasta and grains, there was no significant differences before and during the pandemic (p-value=0.79) (Table 2).\n\nMilk and dairy products group\n\nThe consumption of dairy products was lower during the pandemic; 50.6% of the Lebanese population was consuming dairy products equals to four times and less during the lockdown vs 45.5% before. Moreover, we noticed a remarkable decrease during the lockdown, of 5.1% in the consumption of other dairy products (54.5% before vs 49.4% during; p-value=0.003). As for the milk consumption, no significant differences were stated before and during the pandemic (p-value=0.433) (Table 2).\n\nSugar group (products and beverages)\n\nThe frequency of consumption of sugary beverages and products in the studied population was high before the lockdown; 27.1% and 43.8% of the population consumed sweet snacks (p-value=0.001) and sugary beverages (p-value=0) respectively, five time and more per week (Table 2). However, a decrease of 3% in the consumption of this group was noted during the lockdown (snacks-p-value=0.001; beverages-p-value=0) (Table 2).\n\nFats and oils group\n\nA decrease by 3% in the consumption of fats and oils was observed during the lockdown (82.3% during vs 79.5% before; p-value=0.001). It was also observed that added fats and oils were more frequently consumed in the Lebanese population before the lockdown (20.5% before vs 17.7% during; p-value=0.001) (Table 2).\n\nCalculation of the food consumption score\n\nThe FCS was calculated based on the equation explained previously in the methods. Compared to the period preceding the pandemic, the mean levels and standard deviation (SD) of the FCS in Lebanon was equal to 93.9±41.9 and it decreased to 88.1±44.4 during the pandemic (p-value=0). Additionally, the percentage of people having low FCS has increased during the lockdown from 9.2% to 13.8% (p-value=0). These results are presented in Table 3.\n\nCooking practices and barriers\n\nTable 4 shows that most cooking attitudes and practices (10 out of 13) showed a significant increase in the intra-COVID-19 pandemic. The highest increase was recorded for the response “not having access to foods/ingredients needed or wanted” (36.2% positive answers before lockdown compared to 51.1% during lockdown) (p-value=0). The only practices that showed a significant decrease during lockdown was “throwing away food leftovers” (30.4% before lockdown compared to 26.6% during lockdown) (p-value=0). Yet, a slight decrease was stated during the lockdown, in the attitude of “feeling confident about managing money to buy healthy food” and “feeling confident about cooking a variety of healthy meals” of 0.8% and 0.3% (p-value=0.474 and p-value=0.706) respectively. Moreover, it was observed that the attitudes of “don’t having the funds for the foods/ingredients needed or wanted” and “don't having the access to the facilities needed to cook or bake such as stove, oven, kitchen equipment” increased by 11% and 9.6% during the lockdown (p-value=0 and p-value=0), respectively, despite the null change in the practice of “cooking meals using healthy ingredients” (p-value=0.611). Similarly, a significant increase of around 8.7% and 6.7% was observed in the practice of “cooking with leftover food” and “using other parts of food label to make food choices” (p-value=0 and p-value=0), respectively. More than 66% of people were “changing recipes to make them healthier”, were “thinking about healthy choices when deciding what to eat “and were “planning meals to have a varied diet”. Additionally, 52.9% of people were “using the nutritional information panel (nutritional breakdown of the products) to make food choices “(Table 4).\n\nCriteria for recipe selection\n\nWith regards to the criteria for recipes selection, the lockdown was found to significantly increase the percentage of agreeing responses for all criterions. For instance, during the lockdown, more than 70% of people selected their recipes had few ingredients (73.7%) that are easily available at home (81%) or at store (79.8%), inexpensive (71.8%) or at good price (81.6%), and healthy (77.5%) (Table 5). Before the pandemic, more than half to three quarters of the people living in Lebanon, agreed that they were selecting recipes that were achievable with few ingredients, were available at home or can be easily found at the store, inexpensive to prepare, healthy and cheap. During the lockdown, an increase ranging between 2.3% and 13.1% was observed in these patterns. The selection of recipes that were based on ingredients easily available at the store, was the only decrease and saw a decrease of 1.2% (Table 5).\n\nShopping\n\nShopping practices have also been affected during the lockdown in Lebanon. Indeed, more than 76% of respondents agreed that they search more for cheapest prices before and during the lockdown (Table 5).\n\nDuring the lockdown, respondents showed a significant reduction of 7.3% less going physically to select and buy food, and that they preferred to order their food products online (variation of 3.6%) they also preferred having it delivered to their home rather than being delivered at a seller’s point (4.1%) (p-value=0.008). Regarding places of groceries shopping, there was a significant decrease during the lockdown for all the places except for shopping straight from the farmer/producer. In addition, it is remarkable that respondents had shown a disinterest in buying food at organic/fair trade shops or specialty stores during lockdown (p-value=0) (Table 6).\n\nFood stock\n\nStocking up foods was also affected during the pandemic. Thereby, we observed a huge increase in stocking up pasta, rice or other grains, for flour, and for legumes/pulses (such as beans, lentils, chickpeas: dried or tinned). This increase was less significant for water, potatoes, yeast, milk, bread, vegetables, and meat. On the other hand, ready-made meals, salty snacks, nuts or nut spread, fish (fresh, frozen and canned), sweet snacks, and plant-based drinks were stored in smaller amounts during the lockdown. Furthermore, lockdown did not show any impact on stocking up non-alcoholic drinks, eggs, other dairy products, fruits in any of its forms as well as vegetarian alternatives (Figure 1, Table 7).\n\nDeterminants of food insecurity in the overall population\n\nMany factors affected the FCS of the people living in Lebanon. Before the pandemic, the percentage of people having low FCS was 9.2% and increased to reach 13.8% during the pandemic (Table 3). The determinants of poor dietary diversity indicated by “low FCS” in the overall population, before and during the pandemic, is conditioned by many variables including the gender, age categories, some cooking practices, cooking with leftovers, education, and employment. Prior to the pandemic, and compared to adolescents (18 years), elderly people were 86% less likely to be affected by the decrease in the FCS [OR:0.14; 95% CI (0.01-1.07)]. The cooking practice that affected the FCS of the population studied the most, was meal planning. 55% of people who planned their meals to include a variety of food [OR:0.45; 95% CI (0.31-0.65)] were less affected by the decrease of the FCS compared to those not practicing this pattern. As for the education, it appears that people with a high school diploma [0.3; 95%CI (0.11-0.82)] were 70% less likely to be affected by the decline in the FCS compared to those who attained lower levels of education. During the pandemic, the binary logistic regression analysis showed that the decrease in the FCS doesn’t seriously affect the elderly [OR: 0.17; 95%CI (0.04-0.72)], when compared with adolescents. Similarly, the decrease in the FCS didn’t affect people with a high school diploma [OR:0.26; 95% CI (0.1-0.66)] and a bachelor degree [OR:0.37; 95% CI (0.15-0.92)], when compared to people with a lower educational level. It was noticed that there was no impact of gender, cooking practices (except for planning meals to include a variety of food before the pandemic), cooking or throwing away leftovers, and employment on the FCS, neither before nor during the lockdown (Table 8).\n\n\nDiscussion\n\nThe present study aimed to investigate the changes in food consumption patterns, diet diversity through the assessment of the FCS and food-related behaviors in Lebanon during the lockdown. In the pre-pandemic and intra-pandemic periods, most food groups were consumed in a frequency of equals to four times and less per week. One in two people were consuming fruits and two in five people were consuming vegetables. Moreover, four out of five people were consuming legumes and pulses and four in five people were consuming nuts.\n\nAs for unprocessed fish, poultry, and meats, nine in ten people were consuming these foods and three out of four people were consuming wholewheat bread, pasta, and grains. The consumption of milk and other dairy products were shown in three out of five people, and four in five people were consuming added fats and oils. On the other hand, some food groups were consumed frequently (more than five times per week): 4 in 25 people were consuming processed meat/poultry/fish and vegetarian alternatives, 17 in 50 people consumed sugary products, and 7 out of 20 were consuming refined grains group. The confinement induced an increase in the consumption of legumes and pulses (3.2%, p-value=0.001) and whole wheat groups (2.8%, p-value=0.03). In contrast, a decrease of 5.4%, 6.9%, 5.8%, 5.1%, 3.1%, 3.4% and 2.8% was observed in the consumption of fruits (p-value=0), vegetables (p-value=0), processed meats, poultry, and fish (p-value=0), other dairy products (p-value=0), sweet snacks (p-value=0.001), sugared beverages (p-value=0), fats and oils (p-value=0.001), respectively. The average FCS decreased by 4.6% during the lockdown. Most cooking practices (10 out of 13) showed a significant increase during the lockdown and the proportions of food stocked have been changing since the start of COVID-19 with larger amounts of pasta, rice or other grains, flour, and legumes/pulses being stocked. No such change was observed in the storage of non-alcoholic drinks, eggs, other dairy products, fruits in any of its forms, as well as vegetarian alternatives. Since 2020, Lebanon is facing economic and political turmoil that has no sign of ending presently. In addition, the country is densely populated with 6.9 million people, 87.2% of whom live in urban areas, including 2 million displaced persons and 500,000 migrant workers.9 Households are primarily multi-generational households, with an average of five people per household.10 Several additional challenges were observed throughout the pandemic, such as the absence of food, having to travel to several places to find it, inability to afford some food types, worries on food safety, and finding the best-price shops for buying some foods.\n\nThe preventive measures for COVID-19 (i.e., physical distancing) and the international recommendations concerning restrictions on travel that were imposed by WHO and Lebanese government, along with the fear of having COVID-19 infection, have led to quarantining of millions of people. This has meant that the tourism, the hospitality industries, the food supply chains, the health system and the industry sector have all seen a sharp decline. Moreover, the production and transportation of fresh nutritious food, such as fruits and vegetables, meat, milk, and other dairy products were affected during the pandemic. In most countries, the cheap highly processed, packaged foods characterized by long shelf life, highest amounts of total fats, saturated fats, sugars and/or salt were frequently consumed leading to lower quality diets.1 Upon the exponential increase in COVID-19, with the consequences on the financial status of consumers, food insecurity started to aggravate. Consumers tend to limit the purchase of food types they cannot afford such as meat and fish and start consuming higher quantities of starchy food due to their wide availability and cheap prices.11\n\nFood consumption and dependency on specific food groups may also be related to the curfew consequences of this virus. In other words, the mental status and anxiety related to food availability can push food insecure consumers toward the consumption of more fruits, savory snacks, sweets, and candies which in turn can cause weight gain.12 Furthermore, the COVID-19 pandemic was acquainted by a need for food-based and non-food based coping strategies to compensate for household economic crisis. Internationally, all sources of income were affected which led to a reduction in resources and an increase in food insecurity prevalence rates.1 Consumers were forced to change their food consumption patterns involuntarily, rely on savings, sell durable household assets and livestock, buy foods with a long shelf-life, eat less, buy cheaper food, and accept food from a friend as a mean of food-based coping. Regarding non-food based coping strategies, consumers relied more on savings, obtained credits, sent household members elsewhere, signed up for governmental programs and borrowed money from friends or family.13 Still remaining was the impact of COVID-19 on the access and availability of resources which constitutes the base to start combating food insecurity.\n\nThis pandemic has altered the agriculture sector in Lebanon, food demand, hunger and malnutrition, and world food prices that exacerbated the food security status.14 The FCS is used to assess the dietary diversity and relative food frequency along with the household’s food security. The decrease in the FCS in Lebanon may be related to several factors such as the critical political and economic situations affecting the country.15 Clashes, political conflicts, and civil insecurity are the main driving factor of food insecurity in Lebanon in recent years due to numerous factors such as refugee migration, army conflicts and political instability.6 In this study, other factors were influencing the FCS such as planning meals, education level, and age category. In fact, people who planned their meals and gave many concerns to variety foods, people with a high school diploma and bachelor’s degree, and elderly people were less affected by the decline of FCS. All these factors may be directly related to meal organization abilities, knowledge, economic status, and person responsible for preparing food.\n\nTo mitigate the effects of income losses and decrease in purchasing power, people started to adjust to these shortages through different coping strategies. Among these strategies, a significant change in most cooking practices was observed such as cooking more with leftovers in addition to minimizing food waste. Furthermore, COVID-19 had led people suffer a significant increase in barriers to cooking healthy meals due to the unavailability of money and less accessibility to food and cooking facilities. During the curfew, people searched more for inexpensive recipes that could be achieved with fewer ingredients. This can be explained by the financial struggle communities are having. These struggles also lead to more people preserving food leftovers rather than throwing them away and ensuring enough resources for healthy cooking with a high emphasis on the cultural role of females in cooking.16 Even when purchasing ingredients, people tend to search more for cheaper prices and less organic foods but order them online rather than going physically to the stores.13,17–20 These statements came hand in hand with our findings where the most cooking practices (10 out of 13) showed a significant increase during the lockdown. The highest increase was recorded for not having access to foods/ingredients needed or wanted and the lowest one for throwing away food leftovers. Besides, proportions of food stocked have been changing since the start of COVID-19 and higher amounts of pasta, rice or other grains, flour, and legumes/pulses were stocked with an unchanged consumption rate of non-alcoholic drinks, eggs, other dairy products, fruits in any of its forms as well as vegetarian alternatives. This may be related to the perceived need for these food groups and their cheap prices.\n\nWhen comparing our results with findings from other countries’ and studies, we observed that the home isolation due to the pandemic induced changes in food-related behaviors in Russia, of which a decrease of meat consumption and sweet products was observed in 1047 adults along with an adoption of healthier consumption patterns.21 In addition, an American survey (n=484 adult participants), suggested people facing food insecurity reduced their intake of fruits and vegetables since the start of the pandemic.22 The findings of the American study, as well as our findings, supported two Italian surveys. One of which stated 18% of the respondents reported a decrease in the consuming of fresh fruits while in another study, 8.7% of the respondents reported a decrease in the consumption of fresh fruits and vegetables.23,24 However, a third study conducted in Italy stated that 2768 adults showed an improvement in their diet quality in which they increased their consumption of fruit (24.4%), vegetables (28.5%), legumes (22.1%), nuts (12%), and fish or shellfish (14%). On the other hand, an excessive consumption of sweets or pastries (36.9%) increased in this study.25 A survey by Enriquez-Martinez et al., conducted in Argentina, Brazil, Mexico, Peru, and Spain on 6,325 adults revealed that most Spanish participants (61.6%), didn’t show any improving or worsening in their food pattern.26 People living in Argentina and Brazil reported some amelioration in their diets. Lower change in food consumption patterns was observed among Peruvians and Mexicans (OR: 0.51; 95% CI: 0.4–0.6 and OR: 0.69; 95% CI: 0.4–0.8, respectively), when compared to Argentinians.26 A Polish survey enrolled in Poland (n=2381 adults) shows that 30% of respondents improved their food patterns through a rise in the consumption of vegetable, milk and other dairy products. In addition, the consumption of fast food, and commercial pastry along with confectionary and salty snacks decreased in more than 50% of respondents. Moreover, one-fifth of the respondents reduced the intake of sugar-sweetened beverages and alcohol.27 In France, in comparison to the period preceding the pandemic, the nutritional quality of the respondents (n=938) diets was affected strongly; a sharp increase in processed meat, sweet-tasting beverages, and alcoholic beverage consumption had negatively decreased the overall diet quality.28 In addition, a French web-based survey that encompassed 37,252 adults showed a decline in the intake of fresh food of where 17% of participants reported a decrease in consumption of fresh fruits, 18% reported a decrease in consumption of fresh vegetables, 22% in fresh red meats, and 31% in fresh fish. On the other hand, 22% of people reported an increase in the consumption of sweets and chocolate, 20% in cookies and cakes, and 18% in cheese along with a decline in the consumption of sandwiches, pizzas, or savory pies (17%).29 The Lithuanian COVIDiet Study (n=2447 participants) that revealed frequent and high consumption of sweets, biscuits, cakes, frozen and canned foods and low intake of fruits and vegetables, also reported frequent cooking and eating out of control. However, this was associated with some positive changes such as high consumption of fresh fruits, vegetables, fish, legumes, and white meat and low consumption of processed meat, carbonated and sugary drinks.30 The findings from a survey conducted by three European countries (n=1071 adults): from Poland (n=407), Austria (n=353) and the United Kingdom (n=311) showed a frequent purchase of frozen foods with long shelf life and high consumption of dairy products, grains, fats, vegetables, and sweets (p-value<0.05).31 In Belgium, (n=8640 adults), 10.4% of participants reported food shortages and 10.3 % were incapable of eating a nutritious diet during lockdown. This status of food insecurity was associated with a change in most dietary behaviors.32 The people living in Denmark, Germany, and Slovenia witnessed, during the pandemic, a decline in the intake of fresh food.33 Three studies in Brazil were conducted between 2020 and 2021 in which more than 50,000 adults were interviewed through web-based questionnaires. The first study highlighted the increase in consumption of processed and energy dense foods (potato fries, chocolate, and ice cream) among Brazilian adults.34 In the second study, an increase in the consumption of vegetables, fruits, and legumes along with a steadiness in the consumption of processed foods was observed.35 The third study described the reduction in consumption of fruits and vegetables along with an increase in the consumption of candies and fast-food.36 In Chile (n=700 adults), it was shown that there was a low consumption of legumes and water and high consumption of junk food (e.g., food with low food quality, low contribution of micronutrients and with a high contribution of sugar, saturated fat, and sodium) and fried foods.37 In China (n=2702 adults), many participants didn’t witness any modification in their habitual diet, while 38.2% increased their snack intake, during their home isolation. This could be explained by the fact that basic food supplies were offered by the government in China during the lockdown.38 Similarly, in the Netherlands, 83% of participants did not modify their eating habits during their confinement.39 A scoping review on many populations (US, Asia including Palestine, India, and China, Europe including Italy, France, Spain, Poland, and the UK, Australia, and Zimbabwe) showed an increase in the consumption of meals and snacks, an amelioration in dietary patterns associated with an increase in fresh produce and home cooking and reductions in comfort food and alcohol consumption. However, in the same review, nine studies showed a reduction in fresh produce, with a further six describing an increase in the consumption of comfort foods such as sweets and processed foods. An increase in alcohol consumption was reported in two studies and weight gain was reported in fifteen studies a reduction in physical exercise.40\n\nAt regional level, in Jordan, among a total of 3129 Jordanians, 23.1% of the respondents were facing severe food insecurity, 36.1% were moderately food insecure and 40.7% were food secure. Food insecure people were consuming fewer meats and carbohydrates compared to food secure people.11 In Iran, the household’s dietary diversity and food security status were studied and a decline in the consumption of white roots and tubers, dark green leafy vegetables, other fruits, organ meat, legumes, nuts and seeds, sweets, spices, condiments, and beverages was shown.41 In addition, the impact of COVID-19 pandemic on food purchasing and dietary behaviors was studied in three Kuwaiti surveys with 1935 respondents in Kuwait. In the first study, an increase in the consumption of vegetables, fruits, and carbohydrates along with a decrease in the consumption of fish and sugary drinks was observed.42 In the second study, a change in the consumption of only fish and seafood was observed.17 In the third study, 50% of respondents reported no changes at all in the consumption of all food groups (44%). In addition, more than half the participants ate more fruits and vegetables, 41.5% ate more legumes and pulses, and 2.3% ate fewer fast food.18 In Lebanon, more than 35% of people did not eat fruits and vegetables daily. In addition, 28% consumed sweets or desserts and 24% drink sweet beverages at least once per day and 30.9% consumed salty snacks (nuts, crackers, chips).19 Moreover, according to Hoteit et al., 9 in every 16 households ate less than one or two meals daily and the majority of them were omitting their meals to spare food. In addition, more than 50% of the Lebanese population were having a low food consumption score.15 Another study conducted in three countries of which Lebanon was included, showed that 33% of people were shopping for their food once per week and 31% of the respondents stated that they were shopping two to three times a week during the COVID-19 pandemic. One in every four were purchasing food less than once a week. Less than 10% of people living in Lebanon, Tunisia, and Jordan were obtaining food and groceries from supermarkets or food delivery services and was significantly the least reported among the Tunisians.20\n\nSome limitations should be considered when evaluating the results of this study. This is a cross-sectional study of which it included retrospective data on the time before lockdown. Thus, due to the respondents’ memory, the presented eating habits could be biased due to the type of questions asked concerning the food patterns before the pandemic. Another limitation concerns the determination of the portion size. Thus, it was not possible to use home measurements or the photographs of products to assist respondents in determining the food portion size.\n\n\nConclusion\n\nThe results of the study indicated some changes in the food consumption patterns with a decline in the dietary diversity that took place due to the social isolation implemented during the COVID-19 pandemic. The reduction in the frequency of consumption of fruits, vegetables, processed meats, poultry, and fish, dairy products (e.g., cheese), sweet snacks, sugared beverages, and fats and oils was clearly observed in our population. On the other hand, an increase in the consumption of legumes and pulses, and whole wheat group was observed also. In the short term, according to our findings, nutrition behavior and the dietary diversity did change during lockdown. Nevertheless, most cooking practices showed a significant increase during the lockdown in the overall population and the proportions of food stocked have been changing since the start of the pandemic where higher amounts of pasta, rice or other grains, flour, and legumes/pulses were stocked with an unchanged storage rate of non-alcoholic drinks, eggs, dairy products, fruits, and vegetarian alternatives. All these changes in dietary behaviors might lead to a negative health consequence. It is essential to foster the governmental role along with the social programs, non-governmental agencies, and public health interventions in encouraging the revitalization of the Lebanese traditional healthy cuisine.\n\n\nData availability\n\nOSF: CORONACOOKING COVID-19 Lebanon. https://doi.org/10.17605/OSF.IO/PRTDQ.43\n\nThe project contains the following underlying data:\n\n- Lebanon_DataSet.sav (owned by the corresponding authors).\n\nOSF: Supplementary materials CoronaCookingSurvey April 2020 https://doi.org/10.17605/OSF.IO/8GPZX.44\n\nThe project contains the following extended data:\n\n- Corona_Cooking_Survey_English_Food security.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to thank Lauranna Teunissen, Isabelle Cuykx, Paulien Decorte, Sara Pabian, Kathleen Van Royen and Charlotte De Backer for the initiation, design, and coordination of the data collection 9 that was part of a bigger international project. Also to thank Elissa Naim for her assistance in data collection. The authors confirm the approval of the acknowledged persons to be acknowledged.\n\n\nReferences\n\nFAO, IFAD, UNICEF, WFP and WHO: In Brief to The State of Food Security and Nutrition in the World 2020. Transforming food systems for affordable healthy diets. Rome: FAO; 2020. Accessed January 19, 2021. Publisher Full Text\n\nFood Security Information Network (FSIN): 2020 Global report on food crises: Joint analysis for better decisions. Rome, Italy and Washington, DC: Food and Agriculture Organization (FAO); World Food Programme (WFP); and International Food Policy Research Institute (IFPRI); 2020. Reference Source\n\nWHO Coronavirus (COVID-19) Dashboard: September 2, 2021. Reference Source\n\nWHO Coronavirus (COVID-19) Dashboard: September 18, 2021. Reference Source\n\n2020 State of Food Security and Nutrition in the World report: Rising hunger and COVID-19 present formidable challenges. International Food Policy Research Institute. Accessed on January 30. Reference Source\n\nArab Horizon: Prospects for Enhancing Food Security in the Arab Region. FAO; 2017; vol. 2030. .\n\nFAO, IFAD, UNICEF, WFP, WHO and ESCWA: Regional Overview of Food Security and Nutrition in the Near East and North Africa 2020 - Enhancing resilience of food systems in the Arab States. Cairo: FAO; 2021. Publisher Full Text\n\nWFP Lebanon Country Brief: World Food Program.August 2021. Reference Source\n\nDe Backer C, Teunissen L, Cuykx I, et al.: An Evaluation of the COVID-19 Pandemic and Perceived Social Distancing Policies in Relation to Planning, Selecting, and Preparing Healthy Meals: An Observational Study in 38 Countries Worldwide. Front. Nutr. 2021; 7: 621726. PubMed Abstract \| Publisher Full Text\n\nKhoury P, Azar E, Hitti E: COVID-19 Response in Lebanon: Current Experience and Challenges in a Low-Resource Setting. JAMA. 2020; 324(6): 548–549. 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Publisher Full Text\n\nWolfson JA, Ishikawa Y, Hosokawa C, et al.: Gender differences in global estimates of cooking frequency prior to COVID-19. Appetite. 2021; 161: 105117. PubMed Abstract \| Publisher Full Text\n\nHusain W, Ashkanani F: Does COVID-19 change dietary habits and lifestyle behaviours in Kuwait: a community-based cross-sectional study. Environ. Health Prev. Med. 2020; 25(1): 61. PubMed Abstract \| Publisher Full Text\n\nAlTarrah D, AlShami E, AlHamad N, et al.: The Impact of Coronavirus COVID-19 Pandemic on Food Purchasing, Eating Behavior, and Perception of Food Safety in Kuwait. Sustainability. 2021; 13: 8987. Publisher Full Text\n\nCheikh Ismail L, Hashim M, Mohamad MN, et al.: Dietary Habits and Lifestyle During Coronavirus Pandemic Lockdown: Experience From Lebanon. Front. Nutr. 2021; 8: 730425. PubMed Abstract \| Publisher Full Text\n\nFaour-Klingbeil D, Osaili TM, Al-Nabulsi AA, et al.: An on-line survey of the behavioral changes in Lebanon, Jordan and Tunisia during the COVID-19 pandemic related to food shopping, food handling, and hygienic practices. Food Control. 2021; 125: 107934. PubMed Abstract \| Publisher Full Text\n\nHassen TB, El Bilali H, Allahyari MS, et al.: Food purchase and eating behavior during the COVID-19 pandemic: A cross-sectional survey of Russian adults. Appetite. 2021; Volume 165: 105309. 0195-6663. PubMed Abstract \| Publisher Full Text\n\nLitton MM, Beavers AW: The Relationship between Food Security Status and Fruit and Vegetable Intake during the COVID-19 Pandemic. Nutrients. 2021; 13(3): 712. PubMed Abstract \| Publisher Full Text\n\nRenzo LD, Gualtieri P, Pivari F, et al.: Eating Habits and Lifestyle Changes during COVID-19 Lockdown: An Italian Survey. J. Transl. Med. 2020; 18: 1–15. PubMed Abstract \| Publisher Full Text\n\nScarmozzino F, Visioli F: Covid-19 and the Subsequent Lockdown Modified Dietary Habits of Almost Half the Population in an Italian Sample. Foods. 2020; 9: 675. PubMed Abstract \| Publisher Full Text\n\nGrant F, Scalvedi ML, Scognamiglio U, et al.: Eating Habits during the COVID-19 Lockdown in Italy: The Nutritional and Lifestyle Side Effects of the Pandemic. Nutrients. 2021; 13(7): 2279. PubMed Abstract \| Publisher Full Text\n\nEnriquez-Martinez OG, Martins M, Pereira T, et al.: Diet and Lifestyle Changes During the COVID-19 Pandemic in Ibero-American Countries: Argentina, Brazil, Mexico, Peru, and Spain. Front. Nutr. 2021; 8: 671004. PubMed Abstract \| Publisher Full Text\n\nGórnicka M, Drywień ME, Zielinska MA, et al.: Dietary and Lifestyle Changes During COVID-19 and the Subsequent Lockdowns among Polish Adults: A Cross-Sectional Online Survey PLifeCOVID-19 Study. Nutrients. 2020; 12(8): 2324. PubMed Abstract \| Publisher Full Text\n\nMarty L, de Lauzon-Guillain B , Labesse M, et al.: Food choice motives and the nutritional quality of diet during the COVID-19 lockdown in France. Appetite. 2021; 157: 105005. PubMed Abstract \| Publisher Full Text\n\nDeschasaux-Tanguy M, Druesne-Pecollo N, Esseddik Y, et al.: Diet and physical activity during the coronavirus disease 2019 (COVID-19) lockdown (March-May 2020): results from the French NutriNet-Santé cohort study. Am. J. Clin. Nutr. 2021; 113(4): 924–938. PubMed Abstract \| Publisher Full Text\n\nKriaucioniene V, Bagdonaviciene L, Rodríguez-Pérez C, et al.: Associations between Changes in Health Behaviours and Body Weight during the COVID-19 Quarantine in Lithuania: The Lithuanian COVIDiet Study. Nutrients. 2020; 12(10): 3119. PubMed Abstract \| Publisher Full Text\n\nSkotnicka M, Karwowska K, Kłobukowski F, et al.: Dietary Habits before and during the COVID-19 Epidemic in Selected European Countries. Nutrients. 2021; 13(5): 1690. PubMed Abstract \| Publisher Full Text\n\nVandevijvere S, De Ridder K, Drieskens S, et al.: Food insecurity and its association with changes in nutritional habits among adults during the COVID-19 confinement measures in Belgium. Public Health Nutr. 2021; 24(5): 950–956. PubMed Abstract \| Publisher Full Text\n\nJanssen M, Chang B, Hristov H, et al.: Changes in Food Consumption During the COVID-19 Pandemic: Analysis of Consumer Survey Data From the First Lockdown Period in Denmark, Germany, and Slovenia. Front. Nutr. 2021; 8: 635859. PubMed Abstract \| Publisher Full Text\n\nMalta DC, Szwarcwald CL, Barros M, et al.: The COVID-19 Pandemic and changes in adult Brazilian lifestyles: a cross-sectional study, 2020. A pandemia da COVID-19 e as mudanças no estilo de vida dos brasileiros adultos: um estudo transversal, 2020. Epidemiologia e servicos de saude: revista do Sistema Unico de Saude do Brasil. 2020; 29(4): e2020407. PubMed Abstract \| Publisher Full Text\n\nSteele EM, Rauber F, Costa C, et al.: Dietary changes in the NutriNet Brasil cohort during the covid-19 pandemic. Rev. Saude Publica. 2020; 54: 91. PubMed Abstract \| Publisher Full Text\n\nSouza TC, Oliveira LA, Daniel MM, et al.: Lifestyle and eating habits before and during COVID-19 quarantine in Brazil. Public Health Nutr. 2021; 1–11. PubMed Abstract \| Publisher Full Text\n\nReyes-Olavarría D, Latorre-Román PÁ, Guzmán-Guzmán IP, et al.: Positive and Negative Changes in Food Habits, Physical Activity Patterns, and Weight Status during COVID-19 Confinement: Associated Factors in the Chilean Population. Int. J. Environ. Res. Public Health. 2020; 17(15): 5431. PubMed Abstract \| Publisher Full Text\n\nYang GY, Lin XL, Fang AP, et al.: Eating Habits and Lifestyles during the Initial Stage of the COVID-19 Lockdown in China: A Cross-Sectional Study. Nutrients. 2021; 13(3): 970. PubMed Abstract \| Publisher Full Text\n\nPoelman MP, Gillebaart M, Schlinkert C, et al.: Eating behavior and food purchases during the COVID-19 lockdown: A cross-sectional study among adults in the Netherlands. Appetite. 2021; 157: 105002. PubMed Abstract \| Publisher Full Text\n\nBennett G, Young E, Butler I, et al.: The Impact of Lockdown During the COVID-19 Outbreak on Dietary Habits in Various Population Groups: A Scoping Review. Front. Nutr. 2021; 8: 626432. PubMed Abstract \| Publisher Full Text\n\nPakravan-Charvadeh MR, Mohammadi-Nasrabadi F, Gholamrezai S, et al.: The short-term effects of COVID-19 outbreak on dietary diversity and food security status of Iranian households (A case study in Tehran province). J. Clean. Prod. 2021; 281: 124537. PubMed Abstract \| Publisher Full Text\n\nSalman A, Sigodo KO, Al-Ghadban F, et al.: Effects of COVID-19 Lockdown on Physical Activity and Dietary Behaviors in Kuwait: A CrossSectional Study. Nutrients. 2021; 13: 2252. PubMed Abstract \| Publisher Full Text\n\nHoteit M: CORONACOOKING COVID-19 Lebanon.2021, December 13. Publisher Full Text\n\nHoteit M: CoronaCooking Survey International.2022, January 6. Publisher Full Text" }	[ { "id": "121394", "date": "21 Feb 2022", "name": "Nahla Al Bayyari", "expertise": [ "Reviewer Expertise Public health nutrition as well as clinical nutrition surveys and the randomized double blinded clinical trials." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript examine the impact of the COVID-19 pandemic on food consumption patterns and dietary diversity of the Lebanese people. The authors clearly and accurately presented and cited the current literature. The study design was a cross-sectional which is technically appropriate for this type of studies. Sufficient details of methods and analysis were provided and all statistical analysis methods are fit with the study design and variables of the study as well as all interpretations are correct. All data underlying the results available in tables are matched with the results part. The study conclusions were based on the study results which indicated some changes in the food consumption patterns with a decline in the dietary diversity that took place due to the social isolation implemented during the COVID-19 pandemic.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "121391", "date": "22 Feb 2022", "name": "Sabika S. Allehdan", "expertise": [ "Reviewer Expertise Clinical Nutrition and epidemiological nutritional" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI appreciate the opportunity to review this interesting manuscript. I enjoyed reading the manuscript. This study aims to explore food consumption pattern and dietary diversity among Lebanese adults during COVID-19 lockdown. Overall, this is a high quality, clear, concise, and well-written manuscript. This manuscript has great implications for nutrition and public health policy. There are specific comments that should be clarified to improve the scientific quality of this manuscript.\nFirst: Clarify how you calculated the sample size of this study in the methods section.\nSecond: Explain how you calculated ORs and their CIs as well as P-values in the statistical analysis section as you calculated them in table 8.\nThird: In table 1, the first five rows represent data as mean ± SD not as (number) (%). Where are the P-values of t-test and chi-square? It is obvious that mean age of females was significantly lower than mean age of males.\nFourth: Please determine serving (servings or cups or ounces) and timing pattern (daily /weekly) of food groups consumption in table 2 (second column). For example, '4 or less' or '5 or less' servings, cups or ounces per day or week.\nFifth: Food consumption score displayed in table 3 as mean ± SD not as (number) (%) in 2 and 3 rows, so change the name of the last column header in table 3.\nSixth: Insert a legend for each table. Legends should include full name of abbreviation. For example, spell the full name of FCS in the legend of table 3. Identify name of statistical analysis was used to calculate P-value in legend for each 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": [] } ]	1	https://f1000research.com/articles/11-110
https://f1000research.com/articles/10-975/v1	27 Sep 21	{ "type": "Systematic Review", "title": "Prevalence and characteristics of cancer patients with COVID-19: a meta-analysis study", "authors": [ "Johan S. Sitanggang", "Kamal B. Siregar", "Henry H. Sitanggang", "Noverita Sprinse Vinolina", "Henry H. Sitanggang", "Noverita Sprinse Vinolina" ], "abstract": "Background: Cancer patients are considered susceptible to coronavirus disease (COVID-19) due to an immunosuppressive state. This study determined the prevalence of cancer in COVID-19 patients, severe events, case fatality rate, history of anticancer therapy associated with severe events, and type of cancer in cancer patients with COVID-19 in the world. Methods: This study used a meta-analysis study approach, sourcing studies from various countries related to cancer and COVID-19. Inclusion and exclusion criteria were established to select studies. A PRISMA flowchart was presented to assess the selection process. Data from inclusion studies were analyzed using Review Manager 5.4. Results: The prevalence of cancer in COVID-19 patients was 4.63% (95% CI, 3.78-5.49%) worldwide. The lowest prevalence was the Asian study group with 2.36% (95% CI, 1.86-2.87%) and the highest prevalence was the European study group with 10.93% (95% CI, 6.62-15.24%). About 43.26% (95% CI, 34.71-51.80%) of cancer patients with COVID-19 experienced severe events of COVID-19. In total, 58.13% (95% CI, 42.79-73.48%) of cancer patients with COVID-19 who in the last month had a history of anticancer therapy experienced severe events. The prevalence of lung cancer in cancer patients with COVID-19 was 20.23% (95% CI, 7.67-32.78%). Forest plots are also presented related to the results of meta-analysis research. Conclusions: High prevalence of cancer among COVID-19 patients indicates the susceptibility of cancer patients to SARS-CoV-2 infection. Cancer in COVID-19 patients and use of anticancer therapy increase severe events of COVID-19.", "keywords": [ "prevalence", "COVID-19", "cancer", "comorbid", "severe event", "fatality" ], "content": "Introduction\n\nOn December 31, 2019, the World Health Organization (WHO) was notified of cases of pneumonia of unknown cause, which were detected in Wuhan City, Hubei Province, China. From 31 December 2019 to 3 January 2020, a total of 44 pneumonia cases with unknown etiology were reported to the WHO by national authorities in China. The Chinese Centers for Disease Control and Prevention identified a new strain of coronavirus, namely Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), with the name of disease given as Coronavirus Disease 2019 (COVID-19)1.\n\nConfirmed cases of COVID-19 are continually increasing in the world. On January 30, 2020, WHO designated COVID-19 as a Public Health Emergency of International Concern2. Approximately 197,788,117 cumulative cases of COVID-19 had been confirmed and 4,219,578 cumulative deaths had been caused by the COVID-19 disease as of August 3, 20213.\n\nThe existence of the COVID-19 pandemic also affects and increases various risks in individuals with chronic diseases. Of the 1,590 cases of COVID-19 in 575 hospitals in 31 provinces of China, 399 cases were reported to have comorbid diseases. The most common comorbidity found was hypertension with 269 people (16.9%), followed by cardiovascular and cerebrovascular diseases with 59 (3.7%) and 30 (1.9%), respectively. Meanwhile, cancer was also found in 18 (1.1%) of 1,590 people4. Patients with cancer are more susceptible to infection and may have a higher risk of experiencing a severe event of COVID-19 than individuals without cancer because of their systemic immunosuppressive states caused by malignancies and anticancer treatments, such as chemotherapy or surgery5. The severe event in this study was defined as the condition of patients with severe symptoms, patients admitted to the intensive care unit, patients requiring ventilation, or patient death.\n\nTherefore, the existence of a meta-analysis study which in principle combines the results of research from various countries around the world, could make epidemiological assessments of the prevalence of cancer in COVID-19 patients more accurate. In addition, this meta-analysis study also explained the latest developments based on inclusion studies related to cancer and COVID-19. The prevalence of severe event, death, history of anticancer therapy, and types of cancer in cancer patients with COVID-19 were also included in this study.\n\nUsing the PICO (patient, intervention, comparison, and outcome) principle, the patients in this study are COVID-19 patients and, there is no intervention. The comparison found in this study is the history of using anticancer therapy, and the outcome sought is the prevalence of cancer patients in COVID-19 patients, the prevalence of severe event in cancer patient with COVID-19, case fatality rate of cancer patients with COVID-19, and the prevalence of severe event in cancer patient with the history of using anticancer therapy within one month. The PICO question statement that may be obtained is related to the prevalence of cancer patients in COVID-19 patients, what is the prevalence of the incidence of severity of cancer patients with COVID-19, and the prevalence of cancer and COVID-19 patients who experienced severe events with a history of anticancer therapy, especially in the last one month.\n\n\nMethods\n\nThis research uses a meta-analysis study method to estimate the frequency of clustered diseases, such as prevalence and case fatality rate. The time for conducting the research was four months, from August to November 2020. The total series of processes starting from submitting ethics to accountability for research results at Universitas Sumatera Utara took seven months, from July 2020 to January 2021. The checklist used in this meta-analysis was the PRISMA 2009 Checklist.\n\nThe data extraction was carried out using a piloted form with inclusion and exclusion criteria. The inclusion criteria were studies with COVID-19 patient subjects, number and prevalence of COVID-19 patients who also experienced cancer, and journals were in English (pre-print articles and full peer reviewed) that had been circulating on the internet until October 31, 2020. The exclusion criteria of this study were review articles, comments, research conducted on animals, and research that did not contain information regarding the number and prevalence of COVID-19 patients with cancer.\n\nAn online literature search was conducted, sourcing from Pubmed, Science Direct, Springerlink, and Google Scholar. Medical subject headings (MeSH) words used to form the search strategy were “prevalence” AND (“cancer” OR “malignancy” OR “tumor”) AND (“COVID-19” OR “coronavirus” OR “SARS-CoV-2”). The data retrieved was the name of the first author, year of publication, data on the number, prevalence, and several characteristics of cancer patients in COVID-19 patients based on research that had been circulating on the internet until October 31, 2020.\n\nThere were three reviewers, namely Johan S. Sitanggang, Kamal B. Siregar, and Henry H. Sitanggang, who screened articles for this meta-analysis. Initial screening was carried out by looking at the suitability of the title against the inclusion and exclusion criteria as well as the study abstract. Studies were then assessed in full-text to assess the presence of information related to prevalence that can be retrieved according to the inclusion and exclusion criteria. Information related to the prevalence of cancer patients in COVID-19 patients, the prevalence of cancer patients and COVID-19 who experienced severe events, case fatality rate of cancer patients and COVID-19, prevalence of severe events in cancer patients with a history of using chemotherapy in the last one month, and prevalence of specific cancer (lung cancer) in COVID-19 patients with cancer, was extracted from the full-text study data and recapitulated with table in Review Manager 5.4.\n\nIn the process of selecting and extracting information from the original study, the reviewers also looked at the research methodology of the original study, the confidence interval, and the p-value of each related study to assess the potential bias of individual studies. The method used in relation to the risk of bias accross studies in this prevalence meta-analysis is quantitative assessment of the p value and z test of each prevalence data table. Prevalence rate and case fatality rate data processing in this meta-analysis study was determined whether by random effect or fixed effect by assessing I2. If I2 is more than 50%, it indicates high heterogeneity between studies, so the random effects model is used. Meanwhile, if I2 is less than 50%, then the fixed effects model is used. There is no additional analysis other than what has been described previously.\n\nData processing that would be carried out in this study was a meta-analysis study. Prevalence rate (PR), 95% confidence interval (CI) were analyzed using Review Manager 5.4 software (The Cochrane Collaboration, Oxford, UK). The heterogeneity between the studies was estimated using the I2 test and q.\n\n\nResults and discussions\n\nFrom the results of literature searches up to October 31, 2020 using MeSH words predetermined, 19,045 literatures were found on Google Scholar, Pubmed, Springer Link, and Science Direct. Of the total 19,045 literatures obtained, 16,500 came from a Google Scholar search. Meanwhile, 1,794 literatures came from Science Direct, 558 literatures came from Springer Link, and 193 literatures came from Pubmed. After going through the selection process, in the end, 47 research literatures were included in this study. The process of searching and selecting the literature for this study can be seen in Figure 1.\n\nFlowchart (a. detailed literature tracing and selection flowchart, b. PRISMA flowchart).\n\nThese studies included information regarding the number, prevalence, and characteristics of cancer patients with COVID-19. The characteristics of each study that had been included in this study could be seen in Table 1.\n\nAccording to the origin of the studies, these studies were found to come from several countries which could be divided based on the location of the country on the continent. The majority of studies that were included in this meta-analysis came from Asia with a total of 33 studies. In this study, there were nine studies (19.1% of all studies) originating from America with all studies originating from the United States of America. Fom Europe, there were five studies (10.6%) included in this meta-analysis.\n\n\nMeta-analysis results of cancer prevalence in COVID-19 patients\n\nIn Figure 2, a forest plot for a total of 47 studies that had been included from various regions of the world. Based on this meta-analysis, it had been found that the overall prevalence of cancer patients in COVID-19 patients was 4.63% (95% CI, 3.78-5.49%). As for the heterogeneity test in this meta-analysis, it had been found that the I2 value was 96% (>75%). This indicates a high degree of heterogeneity in the overall study results. Therefore, a meta-analysis was performed with random effect (>50%). High heterogeneity was also indicated by the P value <0.0001 (<0.05) in this study. The result of the P value on the Z-test was <0.0001 (<0.05), which means that the 47 studies’ data had significant and important values.\n\nTherefore, the prevalence of cancer in COVID-19 patients in the world had been found to be eight times higher than the prevalence value of cancer in the whole world population based on the latest WHO data. The prevalence of cancer sufferers in the world community only reaches 0.57%52. The high prevalence of cancer patients in COVID-19 patients shows that cancer sufferers are more susceptible to infection from the SARS-CoV-2 virus, which must be closely monitored.\n\nOn the Asian continent, the results of the meta-analysis of the prevalence of cancer patients in COVID-19 patients was 2.36% (95% CI, 1.86-2.87%). Meanwhile, in the Americas, the results of the meta-analysis of the prevalence of cancer patients in COVID-19 patients was 6.92% (95% CI, 5.92-7.92%).\n\nBased on studies originating from Europe, the results of the meta-analysis of the prevalence of cancer in COVID-19 patients was 10.93% (95% CI, 6.62-15.24%). The prevalence of cancer patients in COVID-19 patients in Europe was the highest compared to the prevalence of the two other continents.\n\nAccording to the studies that had been included, there were reports of cancer patients with COVID-19 experiencing a severe event, and case deaths. The severe event in this study was defined as the condition of patients with severe symptoms, patients admitted to the intensive care unit, patients requiring ventilation, or even death.\n\nFigure 3 presents the prevalence of severe event that occurs in cancer patients with COVID-19. Based on meta-analysis calculations from a total of 26 studies containing information regarding severe event in cancer patients with COVID-19, it was found that the prevalence value was 43.26% (95% CI, 34.71-51.80%). The I2 value was 91% (>75%), so that the calculation of this meta-analysis also used random effect. Heterogeneous P values and P on the Z-test were found to be <0.00001 (<0.05) which was heterogeneous and significant.\n\nBased on the studies originating from China, it was found that the prevalence of severe event in cancer patients with COVID-19 was 49.67% (95% CI, 37.34-62.00%). Meanwhile, according to three other studies originating from Asia outside China, it was found that 67.02% (95% CI, 0.00-100.00%) of cancer patients with COVID-19 experienced severe event. The prevalence of severe event based on studies originating from Asia outside of China had the highest prevalence value among other groups33,34,37.\n\nThe prevalence of severe event based on American studies was the lowest of the other groups. Based on 5 studies from America, 27.99% (95% CI, 12.21-43.76%) cancer patients with COVID-19 experienced severe event. Additionally, it was found that 44.25% (95% CI, 7.64-80.86%) of European cancer patients with COVID-19 experienced severe event.\n\nThe fatality rate of COVID-19 cases in cancer patients was based on 12 studies which were described in detail in Figure 4. The result was a case fatality rate of 26.29% (95% CI, 18.09-34.49%) of cancer patients with COVID-19 who experienced death. Based on the I2 value related to the heterogeneity of the study, it was found that a high level of heterogeneity was obtained with a I2 of 88%. Therefore, the principle of random effect was used in calculating the prevalence meta-analysis. Heterogeneous P values and P on the Z-test were found <0.00001 (<0.05) which was heterogeneous and significant.\n\nIn Figure 5, the prevalence of severe event in cancer patients with COVID-19 with a history of anticancer therapy was described at least in the last month. There were three studies that specifically contained this data. Based on meta-analysis calculations from the three studies, 58.13% (95% CI, 42.79-73.48%) of cancer patients with COVID-19 who in the last month at least had a history of anticancer therapy experienced severe event. The P value on the Z-test was found to be <0.00001, which means that the calculation remained significant and important. In addition to exposure and mobility factors in cancer patients, the state of immunosuppression caused by anticancer therapy in cancer patients is also considered an important factor of susceptibility to COVID-19. The prevalence of the COVID-19 severe event in patients with cancer who had a history of anticancer therapy in the last month was 1.34 times higher than the prevalence of severe event of COVID-19 in cancer patients as a whole.\n\nFigure 6 shows that from five studies that specifically described data on the types of cancer found in cancer patients with COVID-19, lung cancer was found in five studies. The value I2 of the five studies was 74%, which was greater than 50%. So, the principle of random effect was used in the calculation. The P value of heterogeneity was found to be 0.004 (<0.05) and the P value of the Z-test was 0.002 (<0.05), which means that the study data from this calculation was heterogeneous and significant.\n\n\nDiscussion\n\nThis meta-analysis study covered a large area and representative as prevalence and epidemiological data related to cancer and COVID-19, which can be seen in Figure 2. The overall prevalence of cancer patients in COVID-19 patients in the world was 4.63% (95% CI, 3.78% - 5.49%). The highest prevalence of cancer in COVID-19 patients by continent area was found in Europe with 10.93% (95% CI, 6.62% - 15.24%). The highest prevalence of cancer in COVID-19 patients reported by a single study came from the UK, with 24.75% (95% CI, 16.34% – 33.16%)51. Meanwhile, the lowest prevalence was Asia 2.36% (95% CI, 1.86-2.87%). The lowest prevalence by a single study was obtained with a prevalence of 0.50% (0.01% – 0.99%), which was taken from a study from China26.\n\nIn the calculation of the meta-analysis of 47 studies, the I2 value, the P value of heterogeneity, and the P value of the Z-test were also presented. The heterogeneity test of the I2 value in this meta-analysis was found to be 96% (>75%). This indicates that the data from the 47 studies has a high level of heterogeneity. The P value of heterogeneity was also found to be <0.0001 (<0.05) which indicates a high level of heterogeneity and significancy. The P value of the Z-test in this forest plot is <0.0001 (<0.05), which means that 47 studies’ data have significant and important values. The high level of heterogeneity and significance value in this meta-analysis calculation can prove that there is no possibility of bias from the authors on the results of the meta-analysis calculations. In addition, all meta-analysis calculations in this study were found to be meaningful or significant in the results.\n\nCancer in COVID-19 patients and use of anticancer therapy affect severe events of COVID-19 patients. Based on inclusion studies that specifically describe the history of anticancer therapy in cancer patients with COVID-19, we found 58.13% (95% CI, 42.79% – 73.48%) of cancer patients who has COVID-19 and a history of anticancer therapy, experienced a serious event (in Figure 5). This prevalence value is 1.34 times higher than the overall prevalence value of severe event in cancer patients with COVID-19 (43.26%, 95% CI, 34.71% – 51.80%) which can be seen in Figure 3.\n\nSpecifically, this study also includes a meta-analysis study related to the prevalence of certain types of cancer, namely lung cancer (in Figure 6). This is because in several inclusion studies, lung cancer was mentioned as the type of cancer with the highest prevalence compared to other cancers in COVID-19 patients in their study research samples5,6,21,30,41. Based on the calculation of the prevalence of lung cancer in cancer patients with COVID-19, it was found that 20.23% (95% CI, 7.67% – 32.78%) of cancer patients with COVID-19 were lung cancer patients. The I2 value of the five studies was 74%, which was greater than 50%. Indeed, based on these results, the heterogeneity level of the inclusion study was not at the highest level of heterogeneity, but the results were heterogeneous enough to make this meta-analysis calculation using random effects. The P value of heterogeneity was found to be 0.004 (<0.05) and the P value of the Z-test was 0.002 (<0.05), which means that the study data from this calculation was heterogeneous and significant.\n\nThe high prevalence of COVID-19 severe event in cancer patients with a history of anticancer therapy means that anticancer therapy is an important factor in the occurrence of poor outcomes in cancer patients with COVID-19. Therefore, cancer patients who are about to undergo anticancer therapy must be closely monitored so they are not exposed to SARS-CoV-2. In patients with suspected symptoms of COVID-19, it is advisable to consider delaying some anticancer therapies such as chemoterapy, surgery, radiotherapy, and others.\n\nAside from the results reported above, there are several obstacles and shortcomings found in the work of this study. In determining a criteria for a severe event, until now there is still no specific value or scoring criteria that determines how severe a patient's condition is caused by COVID-19.\n\nHigh prevalence of cancer among COVID-19 patients indicates the susceptibility of cancer patients to SARS-CoV-2 infection. Cancer in COVID-19 patients and use of anticancer therapy affect the prevalence of a severe event of COVID-19 patients. The prevalence of severe event in patients with cancer and COVID-19 who had a history of anticancer therapy in the last 1 month was 1.34 times higher than the prevalence of severe event in cancer patients with COVID-19 as a whole. This means that a history of anticancer therapy may influence the occurrence of COVID-19 severity in cancer patients with COVID-19. All authors hope that more specific research about COVID-19 and certain type of cancer in the future will be carried out.\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 ‘Prevalence and characteristics of cancer patients with covid-19: a meta-analysis study’. https://doi.org/10.6084/m9.figshare.1659004453\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nEthical approval\n\nBased on the approval of the health research implementation ethics committee No. 462 / KEP / USU / 2020, Chair of the Research Ethics Committee of the Universitas Sumatera Utara, after carrying out discussion and assessment of research proposals based on the rules of the Neuremberg Code and the Declaration of Helsinki, decided on a study entitled, \"Prevalence and Characteristics of Cancer Patients with COVID-19: a Meta-Analysis Study\", approved for implementation.", "appendix": "Author contributions\n\nJohan S. Sitanggang: Conceptualization, methodology, software, validation, formal analysis, investigation, data curation, writing - original draft¸ writing - review & editing.\n\nKamal B. Siregar: Methodology, validation, formal analysis, investigation, data curation, supervision, project administration, funding acquisition.\n\nHenry H. Sitanggang: Methodology, validation, formal analysis, investigation, data curation, visualization.\n\nNoverita S. Vinolina: Methodology, software, validation, project administration, funding acquisition.\n\n\nReferences\n\nWorld Health Organization: Novel Coronavirus (2019-nCoV) Situation Report – 1. 2020; [Accessed 24 April 2020]. Reference Source\n\nWorld Health Organization: COVID-19 Public Health Emergency of International Concern (PHEIC) Global research and innovation forum. 2020; [Accessed 24 April 2021]. Reference Source\n\nWorld Health Organization: COVID-19 Weekly Epidemiological Update. 2021; [Accessed 06 August 2020]. 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Dataset. 2021. http://dx.doi.org/10.6084/m9.figshare.16590044.v1" }	[ { "id": "96847", "date": "01 Nov 2021", "name": "Tjakra Wibawa Manuaba", "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\nThere is only one thing I want to clear up, which is the understanding of the title? Prevalence and characteristics of cancer patients with COVID-19? shouldn’t it be the prevalence of covid-19 in cancer patients and their characteristics? Because looking at the study design - this study is to determine the prevalence of cancer in covid-19 patients, it seems to me that COVID-19 was somehow causing cancer.\n\nWhen actually we were looking at how many cancer patients were infected by covid-19 and how they reacted to the infection and whether certain types of cancer and their treatments influenced the outcomes of covid-19 infection. We understand \"the sample finding\" was looking at patients with COVID-19 infection, and looked from those patients who had cancer, so this created a thought that COVID-19 was directly correlated to cancer in fact was not, they were cancer patients who happened to be infected by COVID-19.\n\nIn general, I feel that this article was good and worth indexing.\nSince I am not a biostatistician and this is a meta-analysis study using available data from other studies. One thing you have to look for is the heterogeneity of the data used. The data have been analysed, and data heterogeneity is not significant (?), so the differences in the study variables are really due to the research hypothesis.\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": "7383", "date": "02 Nov 2021", "name": "Noverita Sprinse", "role": "Author Response", "response": "Dear reviewer, Thank you for the review that had been given to this meta-analysis article. We will immediately improve some of the points that have been described by reviewer as follows. The first is related to the title, which was originally \"Prevalence and Characteristics of Cancer Patients with COVID-19\" to \"The Prevalence of COVID-19 in Cancer Patients and Their Characteristics\". Indeed, our current title may imply the assumption that COVID-19 infection may cause cancer. Therefore, based on suggestions and reviews from reviewer, we made the decision to change the title to \"Prevalence of Cancer as a Comorbid in COVID-19 Patients and Their Characteristics\". In our opinion, as the authors, the title \"COVID-19 Prevalence in Cancer Patients\" does not really suitable with the results of the study because the results and sources of the studies used is the prevalence of cancer as a comorbid in COVID-19 patients, which affects the severity of the patients' COVID-19 conditions. Then, regarding the heterogeneity and significance of each data, we have conducted heterogeneity and significance tests for each meta-analytical calculation and studies included in this article. In each meta-analysis calculation and included studies, we use the p-value test, Z-score test, and also the I2 test with the results showing heterogeneous and significant, which we have included the results in the discussions column and can also be seen directly in the meta-analysis calculation figure at the bottom left. Thank you for the time and thoughts that have been given by reviewer to review this article. Best regards, Authors" } ] } ]	1	https://f1000research.com/articles/10-975
https://f1000research.com/articles/11-106/v1	27 Jan 22	{ "type": "Brief Report", "title": "DNA barcoding for the discrimination of Uncaria gambir and its closely related species using internal transcribed spacer genes", "authors": [ "Epi Supri Wardi", "Sumaryati Syukur", "Zulkarnain Chaidir", "Jamsari Jamsari", "Bastian Nova", "Epi Supri Wardi", "Zulkarnain Chaidir", "Jamsari Jamsari", "Bastian Nova" ], "abstract": "Background: Uncaria gambir is one Uncaria species that exclusively grows in Indonesia. The phytochemical constituents of this species have been widely explored and its extracts are used as traditional medicine. However, the relationship between Uncaria gambir and other Uncaria species is still unknown. DNA barcoding was used in this study to reveal this relationship. Methods: Genomic DNA was isolated from four main cultivated variants of Uncaria gambir species in Indonesia. ITS primer was used to amplify the specific gene region. Genetic distance analysis was carried out on Uncaria gambir and 12 other Uncaria species. A phylogenetic tree was created to determine the relationship among Uncaria species using the maximum likelihood method. Results: The ITS primer successfully amplified the ITS region in Uncaria gambir. Genetic distance and phylogenetic tree analyses showed that Uncaria gambir has a close relationship with Uncaria scandens, Uncaria yunnanensis, and Uncaria macrophylla which is also indicated by Interspecific distance analysis. Conclusions: Although the DNA barcoding gap is absent in genetic distance analysis, the phylogenetic tree analysis from the ITS region can differentiate Uncaria gambir from other Uncaria species.", "keywords": [ "Uncaria Gambir", "ITS", "Phylogenetic analysis", "Genetic distance" ], "content": "Introduction\n\nGambier, extracted from the twigs and leaves of Uncaria gambir, is traditionally used to treat various diseases in Indonesia, such as diarrhea, gastrointestinal diseases, burns, acne, and cancer (Musdja et al., 2018). Uncaria gambir is commonly distributed in southeast Asia tropical regions, mostly in Indonesia and Malaysia (Taniguchi et al., 2007). The Sumatera Barat region has become the biggest contributor of gambier with Udang, Cubadak, Riau, and Mancik being the main cultivated variants in Indonesia.\n\nThe identification of Uncaria gambir is still based on morphological characteristics and phytochemical constituents. These identifications are mainly affected by environmental conditions and conditions of the sample, thus resulting in a biased interpretation (Zhu et al., 2011). To address this problem, DNA barcoding can be a solution to discriminate the species by using short and standardized DNA fragments (Li et al., 2021). The Consortium for the Barcode of Life (CBOL) has approved chloroplast genes sequences, such as psbA–trnH, trnL–trnF, ycf1, and rpoC1, as potential DNA barcodes and suggests a combination of matK and rbcL genes as an alternative barcode for Embryophyta (Hollingsworth et al., 2009). In addition, internal transcribed spacer (ITS) genes (ribosomal region) have been proposed as supplemental barcodes for matK and rbcL (Hollingsworth et al., 2009).\n\nThe ITS gene is located between 18S and 28S genes in the nrDNA repeat unit and covers the ITS1 and ITS2 regions, connected by the 5.8S rRNA gene (Bellemain et al., 2010). Previous studies have used restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) to investigate catechin molecular markers in Uncaria gambir (Istino et al., 2013).\n\nMolecular identification using matK and rbcL genes gives a low amplification success rate and was inaccurate to discriminate among Uncaria gambir variants (Wardi et al., 2020). In a recent study, utilization of the ITS-2 gene only gives one species-specific site among Uncaria gambir variants and no genetic distance analysis was studied (Wardi et al., 2021b).\n\nIn this study, we investigated the utilization of the ITS gene to discriminate Uncaria gambir within the other 12 Uncaria species. Genetic distance analysis was carried out to provide relationship data between Uncaria gambir and the other 12 Uncaria species. This study is the first to provide an ITS gene sequence from Uncaria gambir.\n\n\nMethods\n\nSix fresh leaves of four Uncaria gambir variants were collected from Siguntur, West Sumatera Province, Indonesia (1°05'36.8\"S 100°28'27.0\"E -1.093562, 100.474153). Morphology identification was conducted in the Herbarium of Andalas University. Harvested fresh leaves were frozen immediately in liquid nitrogen and was stored at -80°C for 24 hours prior to DNA isolation.\n\nDNA isolation was performed using the CTAB method (Allen et al., 2006). In total 300 mg of frozen leaf sample was ground and put into a 2 ml Eppendorf tube. 1 ml of 2x CTAB buffer and merchaptoethanol was added and vortexed until homogenized. The solution was incubated at 65°C for 30 minutes and inverted every 10 minutes. 500 µL of phenol: chloroform: isoamylalcohol mixture (25:24:1) was added and vortexed for 1 minute then centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a new 2 ml Eppendorf tube. 500 µL of chloroform mixture: isoamylalcohol (24:1) was added and vortexed for 10 minutes then centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a new 1.5 mL Eppendorf tube. Sodium acetate was added as much as 1/10 times the volume of the supernatant, then 1 ml of cold ethanol 99% was added and swirled for 1 minute. The solution was centrifuged at 12,000 rpm for 5 minutes, supernatant was removed and 500 µL of 70% ethanol was added. The solution was centrifuged at 12,000 rpm for 5 minutes, the supernatant was discarded and the DNA was dried in the oven. Finally 100 µL of 1xTE buffer was added to dried DNA and stored at -20°C.\n\nDNA amplification was performed using MyTaqTM HS Red Mix Bioline kit. The final concentrations for a typical 25 μl reaction were as follows: 12.5 µL MyTaqTM HS Red Mix Bioline, 1 µL reverse primer ITS-u4 (RGTTTCTTTTCCTCCGCTTA), 1 µL forward primer ITS-u1 (GGAAGKARAAGTCGTAACAAGG), 1 µL DNA template, 9.5 µL Nuclease-Free Water to obtain a 25 µL final volume. The thermal cycling conditions (BIO-RAD C1000TM Thermal Cycler) were as follows: (a) Denaturation 96°C for 60 s, (b) Denaturation 96°C for 15 s, (c) Annealing 70°C for 45 s (-1°C decrement each cycle), (d) Extension 72°C for 60 s; (e) repeat step (b-d) for 25 cycles and followed by a final extension at 72 °C for 5 min.\n\nThe isolation and amplification results were visualized using electrophoresis with 1.5% agarose gel. The expected amplification product was sequenced by 1st BASE DNA Sequencing Service (Apical Scientific Sdn Bhd) bidirectionally using the specific primer.\n\nA total of 132 Uncaria ITS sequences were downloaded from National Center for Biotechnology Information (NCBI) and were aligned with Uncaria gambir using MEGA X software version 10.2.6 (Tamura et al., 2013). The intraspecific and interspecific distances were computed with Kimura 2-Parameter (K2P) using MEGA X software (Kimura, 1980)(Meier et al., 2006).\n\nThe sequences were analyzed by the maximum likelihood method for phylogenetic tree construction. The maximum likelihood analyses, including 1000 nonparametric bootstrap replicates, was performed using MEGA X software under the Kimura 2-parameter (K2P)+G model. Nauclea orientalis and Nauclea diderrichi sequences that are relative to the Uncaria genus were downloaded from NCBI and used as an outgroup species.\n\n\nResults\n\nThe primer ITS-u1 and ITS-u4 showed 100% amplification efficiency to Uncaria gambir. Sequences were deposited in the NCBI GenBank database under the accession numbers MZ927014, MZ927015, MZ927016, and MZ926993 (Table 1). The region's length was obtained from 644 to 646 bp.\n\nThe dataset used in this study includes 4 species of Uncaria gambir and 132 Uncaria sequences of the ITS region downloaded from the NCBI GenBank database (Extended data). This dataset contained 655 conserve sites, 68 variable sites, and 54 parsimony informative sites.\n\nThe mean of intraspecific distance (Table 2) and interspecific distance (Table 3) is 0.001% and 0.022%, respectively. The BLAST showed the Uncaria gambir Riau, Mancik, Udang, and Cubadak variant had 99.22%, 99.23%, and 99.07% similarity with U. macrophylla MF033304, respectively. In addition, the Uncaria gambir Cubadak variant had 99.22% similarity with U. macrophylla MF033303.\n\nThe phylogenetic tree was constructed using 13 Uncaria species. Uncaria gambir were clustered from other species (Figure 1). The four variants of Uncaria gambir had a close relationship with Uncaria macrophylla MF033303.\n\nThe phylogenetic tree showed Uncaria gambir has a close relationship with Uncaria yunnanensis but not Uncaria scandens. In contrast, the interspecific distance value showed Uncaria gambir had a close relationship with Uncaria yunnanensis and Uncaria scandens.\n\n\nDiscussion\n\nUncaria is a member of the Rubiaceae family and consists of 34 species (Ridsdale, 1978) that are well known as medicinal plants (Heitzman et al., 2005). The method to identify Uncaria gambir is currently limited to morphological identification. This study compares the ITS gene sequence of the four main cultivated variants of Uncaria gambir from Indonesia with 132 other Uncaria sequences (12 species) downloaded from NCBI.\n\nThe interspecific distance analysis showed that Uncaria gambir is significantly different from the other 12 Uncaria species. Uncaria scandens and Uncaria sinensis species had the lowest and highest interspecific distance value, respectively. The intraspecific distance analysis showed Uncaria rhynchophylla, Uncaria rhynchophylloides, Uncaria sessilifructus, and Uncaria macrophylla had a value of 0.001. The intraspecific and interspecific values showed a partial overlap value, thus no gap between the species. Tang et al. (2016) showed similar results when comparing Uncaria rhynchophylla (Miq.) Jacks with 28 other Uncaria species sequences.\n\nIt is important in DNA barcoding to have a gap, as it can differentiate unknown from known sequences, as no DNA barcoding gaps may give inaccurate results in differentiating the species (Liu et al., 2014; Rach et al., 2008). The phylogenetic tree was created with the maximum likelihood method. The advantage of this method is that it includes evolutionary models to account for the variation in the sequences compared with maximum parsimony and distance methods (Mount, 2008).\n\nThe phylogenetic tree showed all of Uncaria gambir species were clustered as monophyletic. All Uncaria gambir variants were nested with Uncaria macrophylla (MF033303), Uncaria lanosa, and Uncaria yunnanensis.\n\n\nConclusion\n\nIn conclusion, the phylogenetic tree ITS sequence can differentiate Uncaria gambir from other Uncaria species. In contrast, genetic distance analysis showed different results as the absence of a DNA barcoding gap means it is not reliable to differentiate between species.\n\n\nData availability\n\nThe ITS sequences from this research are available from NCBI:\n\nGenBank: Uncaria gambir isolate Udang, Accession number MZ927014: https://www.ncbi.nlm.nih.gov/nuccore/MZ927014\n\nGenBank: Uncaria gambir isolate Mancik, Accession number MZ927015: https://www.ncbi.nlm.nih.gov/nuccore/MZ927015\n\nGenBank: Uncaria gambir isolate Riau, Accession number MZ927016: https://www.ncbi.nlm.nih.gov/nuccore/MZ927016\n\nGenBank: Uncaria gambir isolate Cubadak, Accession number MZ926993: https://www.ncbi.nlm.nih.gov/nuccore/MZ926993\n\nDryad: GenBank accession numbers of downloaded sequences used in this study https://doi.org/10.5061/dryad.dfn2z352w (Wardi et al., 2021a)\n\nThis project contains the following extended data:\n\nTable_E1_GeneBank_Accession_Numbers.docx (This data contains accession numbers from 132 sequence of Uncaria species, and two accession numbers from Nauclea).\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\nAllen GC, Flores-Vergara MA, Krasynanski S, et al.: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc. 2006; 1(5): 2320–2325. PubMed Abstract \| Publisher Full Text\n\nBellemain E, Carlsen T, Brochmann C, et al.: ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. BMC Microbiol. 2010; 10(1): 189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeitzman ME, Neto CC, Winiarz E, et al.: Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae). Phytochemistry. 2005; 66(1): 5–29. PubMed Abstract \| Publisher Full Text\n\nHollingsworth PMForrest LLSpouge JLCBOL Plant Working Group: A DNA barcode for land plants. Proc Natl Acad Sci U S A. 2009; 106(31): 12794–12797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIstino F, Hamda F, Irfan S, et al.: Characterization of specific random amplified polymorphic (RAPD) DNA fragments related to catechin content for early detection methods in gambier plant (Uncaria gambir (Hunter) Roxb.). Afr J Biotechnol. 2013; 12(15): 1736–1744. Publisher Full Text\n\nKimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16(2): 111–120. PubMed Abstract \| Publisher Full Text\n\nLi H, Xiao W, Tong T, et al.: The specific DNA barcodes based on chloroplast genes for species identification of Orchidaceae plants. Sci Rep. 2021; 11(1): 1424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu XF, Yang CH, Han HL, et al.: Identifying species of moths (Lepidoptera) from Baihua Mountain, Beijing, China, using DNA barcodes. Ecol Evol. 2014; 4(12): 2472–2487. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeier R, Shiyang K, Vaidya G, et al.: DNA Barcoding and Taxonomy in Diptera: A Tale of High Intraspecific Variability and Low Identification Success. Syst Biol. Edited by M. Hedin, 2006; 55(5): 715–728. PubMed Abstract \| Publisher Full Text\n\nMount DW: Choosing a Method for Phylogenetic Prediction. CSH Protoc. 2008; 2008(5): pdb.ip49. PubMed Abstract \| Publisher Full Text\n\nMusdja MY, Rahman HA, Hasan D: Antioxidant Activity Of Catechins Isolate Of Uncaria Gambier Roxb In Male Rats. LIFE: International Journal of Health and Life-Sciences. 2018; 4(2): 34–46. Publisher Full Text\n\nRach J, Desalle R, Sarkar IN, et al.: Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata. Proc Biol Sci. 2008; 275(1632): 237–247. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRidsdale CE: A revision of Mitragyna and Uncaria (Rubiaceae). Blumea: Biodiversity, Evolution and Biogeography of Plants. 1978; 24(1): 43–100. Reference Source\n\nTamura K, Stecher G, Peterson D, et al.: MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol. 2013; 30(12): 2725–2729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTang YL, Wu YS, Huang RS, et al.: Molecular identification of Uncaria (Gouteng) through DNA barcoding. Chin Med. 2016; 11(1): 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaniguchi S, Kuroda K, Doi KI, et al.: [Evaluation of Gambir Quality Based on Quantitative Analysis of Polyphenolic Constituents]. Yakugaku Zasshi. 2007; 127(8): 1291–1300. PubMed Abstract \| Publisher Full Text\n\nWardi ES, Jamsari J, Irwandi I, et al.: Barkod DNA Pada Tanaman Gambir (Uncaria gambir(Hunter) Roxb.) Berdasarkan Gen matK Dan rbcL. Jurnal Ilmiah As-Syifaa. 2020; 12(1): 22–28. Publisher Full Text\n\nWardi ES, Syukur S, Chaidir Z, et al.: DNA barcoding of Uncaria Gambir using Internal Transcribed Spacer (ITS) Marker. 2021a. http://www.doi.org/10.5061/dryad.dfn2z352w\n\nWardi ES, Syukur S, Chaidir Z, et al.: Molecular identification of Uncaria gambir [Hunter] Roxb. through DNA barcoding. IOP Conf Ser: Earth Environ Sci. 2021b; 741(1): 012056. Publisher Full Text\n\nZhu S, Zhou L, Pang H, et al.: [Molecular identification of one Uncaria plant]. Zhongguo Zhong Yao Za Zhi. 2011; 36(5): 535–7. PubMed Abstract" }	[ { "id": "141126", "date": "20 Jun 2022", "name": "Jarrett Phillips", "expertise": [ "Reviewer Expertise Bioinformatics", "data science", "DNA barcoding", "machine learning", "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\nOverall, it is my opinion that the present short report is partially scientifically sound. However, further analyses needs to be completed to more easily support overall findings.\n\nSince the success of DNA barcoding relies heavily on the barcode gap, analyses demonstrating the presence or absence of a gap between maximum intraspecific distance and minimum interspecific (i.e., nearest neighbour) distance should be done. This includes dotplots and histograms. There is much in the barcoding literature regarding these visual summaries. The Barcode of Life Data Systems (BOLD) (http://v4.boldsystems.org) is a good place to start with this.\n\nAdditionally, only mean distance values are reported and the barcode gap is assessed on this basis. This is not correct. The mean has been shown to over exaggerate the presence of a barcode gap. See Meier et al. (2008)1 for details.\nI would also encourage you to download relevant Uncaria species barcodes from BOLD to supplement the GenBank data. This will help paint a better overall picture within your paper.\nThe barcode gap is highly sensitive to both the depth and breadth of taxon sampling. Sufficient numbers of specimens need to be collected from across known species' geographic ranges. This should be highlighted in the paper.\nThere was no mention of BLAST in the manuscript except in the Results. Clearly, a nucleotide BLAST (blastn) was performed, but there was really no mention of details on this aspect. Please add more detail in this respect. It would also be ideal to run the sequences though the BOLD Identification Engine to support findings. BOLD employs global alignment whereas BLAST uses only local alignment to assess overall similarity matches.\nMaximum likelihood (ML) with the Kimura-2-Parameter (K2P) model was used for phylogenetic analysis instead of the more common neighbour joining (NJ) method. This was explained because ML allows inclusion of the model of nucleotide substitution. However, no formal comparison of other models was given. Typically one chooses the most parsimonious model on the basis of a model selection criterion scubas as the Akaike Information Criterion (AIC) or the Bayesian Information Criterion (BIC). I strongly suggest that this is included in the next manuscript version.\nOnce the above comments are thoroughly addressed, the paper should be in a better place to warrant indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": [] }, { "id": "156757", "date": "08 Dec 2022", "name": "Andrey Yurkov", "expertise": [ "Reviewer Expertise Fungal taxonomy and systematics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 provides an interesting view on molecular recognition of plant species using DNA-barcoding. It has been previously emphasised that species recognitions approaches and the results depend on the used species concepts. Thus, I recommend to introduce in the introduction the presently used species concept (or concepts) of the genus Uncaria. This will help readers to better understand the results of this work.\nI have two critical points:\n1. The results of the phylogenetic analysis should be presented as a distance-based tree or network and not as a cladogram. I also suggest to include statistical support for clades. My feeling is that a phylogenetic network (a distance or parsimony network) might be more suitable for the research question.\n2. The conclusions need to be adjusted to reflect the results, i.e. (i) in the absence of a barcoding gap, one cannot distinguish one species from another; (ii) the number of differences (substitutions and gaps) is technically sufficient to identify Uncaria gambir; and (iii) if this species is really an independent species (e.g. clade support).\nDepending on answers in the second point, I recommend authors to decide on whether they suggest that (i) Uncaria gambir is not an independent species or (ii) that ITS is not a good marker for Uncaria.\nI wish you good luck with your submission.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/11-106
https://f1000research.com/articles/11-105/v1	27 Jan 22	{ "type": "Systematic Review", "title": "Accuracy of radiographic and protrusive occlusal record methods in determining condylar guidance angles: a systematic review and meta-analysis", "authors": [ "Alexander Maniangat Luke", "Sam Thomas Kuriadom", "Jeny Mary George", "Dian Agustin Wahjuningrum", "Sam Thomas Kuriadom", "Jeny Mary George" ], "abstract": "Background: The objective of this systematic review was to compare the accuracy of radiographic and protrusive occlusal record (POR) methods in determining horizontal condylar guidance (HCG) angles in dentate and edentulous patients. Methods: Studies assessing condylar guiding angles in dentulous/partially edentulous and totally edentulous patients free of temporomandibular disorders using both radiographic and protrusive occlusal record methods were included. A comprehensive search with PubMed/MEDLINE, Cochrane Central Register of Controlled Trials, Web of Science, Google Scholar and Open Grey databases was done. Two reviewers extracted the data after eligibility assessment. Study quality was examined using the NIH quality assessment tool and graded based on tooth selection, number of root canals assessed, study environment, number of observers, test reliability report, validation approach, validation criteria, and validity reliability. A meta-analysis of pooled data, subgroups and sensitivity analysis was performed using RevMan (P<0.05). Results: The qualitative synthesis contained 33 papers, 32 of which were included in the meta-analysis. The standardised mean difference between the radiographic and protrusive occlusal record methods for right and left HCG angle in dentate patients was 0.68 [0.37, 0.98] and 0.63 [0.32, 0.95], respectively, and for right and left HCG angle in edentulous patients was 0.80 [0.36, 1.24] and 0.66 [0.18, 1.15], indicating a statistically significant difference (p<0.05). Conclusions: Clinical variability among the selected studies could not be completely avoided and the sample sizes were limited, resulting in a lack of statistical power. To rule out potential causes of heterogeneity, subgroup and sensitivity analyses were done separately for dentate and edentulous individuals for the right and left HCG angle. The present systematic review and meta-analysis concluded that for the dentate and edentulous patients, the right and left HCG angle values determined by radiographic method showed statistically significant difference as compared to the protrusive occlusal records. PROSPERO registration: CRD42020206599 (28/09/2020)", "keywords": [ "Horizontal condylar guidance", "Cone beam computed tomography", "Lateral cephalogram", "Panoramic radiograph", "Protrusive occlusal record" ], "content": "Introduction\n\nThe registration of precise condylar path and mandibular movement on an articulator is critical to the achievement of an adequate oral rehabilitation of the patient. The inclination of the condylar trail, which is one of the five aspects of balanced occlusion, is therefore a crucial factor in prosthetic treatment success.1,2 This is the only aspect that is not under the control of a prosthodontist and should be reproduced precisely. Condylar guidance (CG) is described by the Glossary of Prosthodontic Terms 9 as mandibular guidance created by the condyle and articular disc crossing the shape of the articular eminence (AE).3 Horizontal condylar guidance (HCG) and lateral condylar guidance (LCG) are the two types of CG. The horizontal condylar route is the path of movement of the condyle-disk assembly in the joint cavity during a protrusive mandibular movement, whereas the lateral condylar path is the path of movement of the condyle-disk assembly in the joint cavity during a lateral mandibular movement.4 Ignorantly recorded condylar guidance will result in occlusal interferences during functional actions, increasing chair side time for prosthesis adjustment, which can be unpleasant for both the patient and the prosthodontist.5,6\n\nNumerous strategies, such as interocclusal records, pantographic tracings, electronic jaw tracking devices, radiographic methods, and so on, can be used to determine horizontal condylar inclination, but programming a semi-adjustable articulator with a protrusive interocclusal record after training the patient to trace a gothic arch is still the most commonly used method in clinical practice.1 The accuracy of Gothic arch recording is influenced by factors such as the patient's neuromuscular control, the stability of the record base, and the recording media. Additionally, when the patient moves the jaw laterally during protrusion, the registration of the condylar route alters.7 Even semi-adjustable articulator setup with interocclusal records has a limited degree of repeatability and is susceptible to instrument, operator, and interocclusal record material factors.1\n\nRadiographs can indeed show the shape of the articular eminence and the glenoid fossa of the temporal bone.8 Magnetic resonance imaging (MRI), cone-beam computed tomography (CBCT), computed tomography (CT), panoramic radiograph (OPG), and lateral cephalogram (LC) are currently accessible to visualise temporomandibular joint components.9 The primary advantages of these approaches are that measurements are based on stable bone landmarks and that mistakes caused by operator inexperience and insufficient neuromuscular control of the patient may be prevented.10,11\n\nPanoramic radiography produces a two-dimensional (2D) image of the temporomandibular joint (TMJ) with a flat reflection of the curved surface of the maxilla and a composite image of the tissues in the X-ray's route but it is frequently inaccurate for measuring HCG due to multiple structures being superimposed,9 whereas 3D multiplanar sections acquired from a CBCT scan give a better anatomic perspective of the condyle and its route without the superimpositions shown in 2D radiography images.11,12 Magnetic resonance imaging (MRI) is a thorough examination that is considered in the literature to be the “gold standard” in diagnosing TMJ disorders; nevertheless, it is not often utilised in everyday dentistry practice due to financial constraints.9\n\nThe primary disadvantages to the widespread use of these radiography techniques are the high cost of equipment, discomfort, and radiation exposure to patients.13 In the study conducted by Tannamala et al,14 a difference of 2-4 degrees in HCG angle between OPG and the protrusive occlusal record was found while in the study by Shreshta et al,13 a difference of 9-10 degrees in HCG angle between CBCT images and the protrusive occlusal record was observed whereas in the study by Das et al11 no significant difference between in HCG angle between CBCT images and the protrusive occlusal record was observed. Additionally, there is no agreement on the findings of several methods for determining the HCG angle and whether approach, clinical or radiographic, offers the most accurate HCG angle readings. There is still no research that have given a complete, quantitative study on which diagnostic reasoning may be based. As a result, the purpose of this systematic review and meta-analysis was to examine and evaluate the accuracy of radiographic and protrusive occlusal record (POR) techniques in estimating horizontal condylar guidance (HCG) angles in dentate and edentulous patients.\n\n\nMethods\n\nOur systematic review and meta-analysis was registered in PROSPERO (CRD42020206599) on the 28th September 2020, and it was undertaken and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.15,55\n\nIn the Patient, Intervention, Comparison, and Outcome (PICO) design, the following focused question was proposed: “Is there a difference in the accuracy of radiographic and protrusive occlusal record techniques in estimating condylar guiding angles in dentate and edentulous patients?”\n\nTo obtain papers in the English language, a complete electronic search was conducted the databases PubMed/ MEDLINE, Cochrane Central Register of Controlled Trials, and Web of Science from 1st January 2011 till 31st December 2020. The distinct electronic search of the journals listed in Table 1 was carried out. Google Scholar, Greylit, and OpenGrey were used to conduct searches in the clinical trials database, cross-referencing, and Grey literature.\n\nFor searching articles, Medical Subject Headings (MeSH) terms, keywords, and other free phrases were coupled using Boolean operators (OR, AND). Following the syntactic guidelines of each database, the same terms were utilised across all search platforms. Table 1 shows the search method as well as the population, interventions, comparisons, outcomes, and study design (PICOS) tool.\n\nPopulation (P): Condylar guiding angles were determined in dentulous/partially edentulous and totally edentulous patients who were free of temporomandibular disorders. Dentulous patients with almost complete set of teeth or with partial edentulousness and nearly ideal occlusion with Class 1 molar relationship and aged 18 years and above, as well as completely edentulous patients with well-formed ridges, good neuromuscular control, adequate inter-maxillary space, and orthognathic jaw relation.\n\nInterventions (I): Studies assessing radiographic techniques like cone beam computed tomography (CBCT), orthopantomogram (OPG), lateral cephalograms, computerized tomography (CT) scan and temporomandibular joint (TMJ) tomogram for determining accurate measurements of condylar inclination.\n\nComparison (C): Studies assessing clinical techniques like protrusive occlusal wax record, for determining accurate measurements of condylar inclination\n\nOutcome (O): Condylar guidance angles using different methods irrespective of the methods of quantifying the outcomes.\n\nStudy design (S): Clinical trials, in vivo studies, randomised controlled trials, non-randomised control trials, quasi experimental investigation, before and after research design, and cohort studies comparing radiographic and clinical techniques.\n\nThe following studies were excluded:\n\n1. Non-clinical studies, in-vitro studies, and animal studies. Furthermore, studies that reported on a single intervention were discarded.\n\n2. Studies on patients with TMJ problems, defective restorations, periodontal disease, excessive attrition, and impaired neuromuscular control.\n\n3. Studies not fully available in the database.\n\n4. Article reporting only abstracts were also excluded.\n\n5. Case series, case reports, reviews, and in vitro research were also eliminated.\n\nTwo review authors (A.M.L. and S.T.K.) conducted the search and screening in accordance with the previously defined procedure. After the initial retrieval, duplicates were removed and the titles and abstract of all the results were screened by 2 authors (A.M.L. and A.M.P). Full text publication articles were retrieved for those articles that met the eligibility criteria. The list of excluded articles at the initial retrieval was crosschecked by all the authors and disagreements were resolved by discussing amongst all. The entire papers were evaluated in the second phase, and papers that did not fulfil the inclusion requirements were unanimously discarded. Cohen's kappa (k) determined the degree of agreement between the two reviewers to be 0.90 for titles and abstracts and 0.92 for full-texts. After discussion, the third author (S.M.) settled the disagreements among the authors/reviewers. Some studies included both ‘dentulous’ and ‘edentulous’ patients. If the findings for the subset of teeth fulfilling the qualifying criteria were available in such studies, that subset of teeth was included in the current review. The study was discarded if it was not possible to split the study findings into two groups. The authors of the listed papers were contacted via email to clarify any concerns or missing data.\n\nThe following data were extracted from the included studies by two independent reviewing authors (D.A.W. and J.M.G.) using pilot tested customised data extraction forms: study identification number, place of study, sample size, age of patient, articulator model, make of machine, radiographic techniques, outcome measures, author’s conclusions.\n\nTo assess the risk of bias and methodological quality of the included articles, a simplified version of the NIH (National Institutes of Health) Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies16 was used, as they reported the results of cross-sectional studies. The assessment “Cannot Determine” (CD) was recorded for the element of the questionnaire for which no information was available in the text. Consistency of studies scoring five or more “Yes” out of eight was rated “Good,” consistency of studies scoring three to five “Yes” was considered “Fair,” and consistency of studies scoring fewer than three “Yes” was labelled “Poor”.17\n\nFor statistical analysis, Review Manager (RevMan) 5.3 was utilised. The pooled results for dichotomous data were presented as relative risks (RRs) at 95 % confidence intervals (CIs), with P<0.05 deemed significant. The I2 test at α = 0.10 was used to measure statistical heterogeneity. For I2>50% and P≤0.10, subgroup analysis was performed. The random-effects model was used when I2 was more than 50%. To determine the stability of the data, sensitivity analysis was performed. Funnel plots were used to detect publication bias in research with more than ten trials for each outcome evaluated.18\n\n\nResults\n\nThe preliminary electronic database search yielded 225 titles (PubMed/MEDLINE and Cochrane library) and 778 titles (Google Scholar); hand scanning the reference lists of the selected studies yielded no further articles. After removing duplicates, there were 563 titles left. Out of these 563 articles, 515 were removed at the initial screening after reading the titles and abstracts. Further to the reviewers' analysis and discussions, 48 papers were chosen for full-text evaluation. Following pre-screening, implementation of the inclusion and exclusion criteria, and maintaining of the PICOS questions, 33 studies remained (10 with inappropriate study design, 3 with inappropriate comparison group, and two studies evaluated lateral condylar guidance) and were included in the qualitative analysis, whereas 32 studies were included in the quantitative synthesis. A flowchart of the search results is represented in Figure 1.\n\nThe general characteristics of 33 studies1,7–9,11,13,14,19–44 are summarized in Table 2. All included studies were unicentric trials published between 2011 and 2020. Typically, 26 studies were conducted in India,1,7,11,13,14,19,20,23–27,29,30,32,34–44 two each in Iran8,28 and Saudi Arabia22,33 and one each in Nepal,21 Korea31 and Poland.9 In total, 20 studies included were conducted among dentate patients having a total sample size of 595 and age ranged from 18–43 years1,19,20,21,25,27,30,32,34,37–40,42,44 while 15 studies included were conducted among edentulous patients having a total sample size of 277 and age ranged from 35–75 years.7–9,11,13,14,19,20,22–24,26,28,29,31,33,35,36,41,43 The ethical approval was obtained in 25 studies1,9,11,13,14,19–26,30–36,38,40–43 while informed consent was obtained in only 20 studies.8,9,11,20–26,30,33,35–38,40–43 The funding information was mentioned in only two studies by Godavarthi et al7 and Naqash et al.33 Out the 33 included studies, only 6 studies,11,25,28,35,37,38 have mentioned about the sample size estimation. The brands and the models of the articulator, OPG, CBCT, CT scan, LC and TMJ tomogram varied according to the studies. Radiographically the HCG angle was measured using OPG1,7–9,14,19–23,26–28,30–32,34–40,42 in 24 studies, seven studies recorded HCG angle with CBCT,8,11,25,31,33,35,43 five studies used LC1,23,24,29,41 and one study each recorded HCG angle with CT scan13 and TMJ tomogram.44 For all of the included studies, the orientation jaw relation was registered utilising face-bow and was transferred on to the semi-adjustable articulator employing mounting jig or extra oral gothic arch tracers, and the protrusive interocclusal record was obtained by instructing the subject to protrude the mandible forward by 6mm. The condylar guidance angle was measured between the Frankfort's horizontal plane and the line formed along the posterior slope of articular eminence (AE), connecting the most concave (highest) point on the glenoid fossa and the most convex (lowest) point on the apical portion of AE in all the included studies. The right and left HCG angle was calculated separately for both the dentate and edentulous patients in all the included studies except for studies Amin et al,20 Jerath S et al,25 Katiyar et al26 and Paul et al1 cumulative value of right and left angle is mentioned.\n\nThe included studies' quality evaluation revealed a wide range of results. Table 3 shows the assessment of the risk of bias for the included studies. Out of the 33 studies, only seven studies were rated as good quality studies,8,11,25,28,35,37,39 whereas the remaining 26 studies were rated as fair quality studies.1,7,9,13,14,19–24,26,27,29–34,36,38,40–44 This fair quality was basically due to no justification of sample size, no assessor blinding and no mention about participation rate and confounder blinding.\n\nA final tally of 32 papers1,7,8,11,13,14,19–44 met the quantitative analysis inclusion criteria. Following that, four independent meta-analyses were done to assess the right and left HCG angle in dentate and edentulous patients.\n\nThe pooled outcomes from 16 studies,8,11,13,14,22–24,26,28,29,31,33,35,36,41,43 with a total sample size of 738 each in the radiographic and POR group, the standardized mean difference (SMD) value for HCG angle using random effect model was 0.68 [0.37, 0.98] and showed a statistically significant difference (P<0.0001) between the radiographic and POR group [Tau2=0.41, Chi2=145.77, I2=86%], (Figure 2). After subgroup analysis was performed using a random-effect model, it was discovered that for the CBCT method,8,11,31,33,35,43 there was a statistically significant difference favouring the radiographic method (SMD, 1.35; 95% CI: 0.41–2.30; P<0.00001) with 93% heterogeneity. For OPG method,8,14,22,23,26,28,31,35,36 radiographic method showed a statistically significant difference as compared to POR method (SMD, 0.52; 95% CI: 0.22–0.83; P=0.0009) with 74% heterogeneity. LC method23,24,29,41 showed no statistically significant difference between the radiographic and POR group (SMD, 0.04; 95% CI: -0.30–0.39; P=0.80) with the heterogeneity of 38%. CT scan method13 showed a statistically significant difference between the radiographic and POR method (SMD, 1.41; 95% CI: 0.50–2.32; P=0.002) (Figure 2).\n\nSD - standard deviation, IV - inverse variance, CI - confidence interval.\n\nThe pooled outcomes from 16 studies,8,11,13,14,22–24,26,28,29,31,33,35,36,41,43 with total sample size of 738 each in the radiographic and POR group, the standardized mean difference (SMD) value for HCG angle using random effect model was 0.63 [0.32, 0.95] and showed a statistically significant difference (P=0.008) between the radiographic and POR group [Tau2=0.43, Chi2=151.64, I2=87%], (Figure 3). After subgroup analysis was performed using a random-effect model, it was discovered that for the CBCT method,8,11,31,33,35,43 there was a statistically significant difference favouring the radiographic method (SMD, 1.29; 95% CI: 0.44–2.13; P=0.03) with 94% heterogeneity. For OPG method,8,14,22,23,26,28,31,35,36 radiographic method showed a statistically significant difference as compared to POR method (SMD, 0.51; 95% CI: 0.14–0.87; P=0.006) with 82% heterogeneity. LC method23,24,29,41 showed no statistically significant difference between the radiographic and POR group (SMD, -0.05; 95% CI: -0.54–0.44; P=0.84) with the heterogeneity of 67%. CT scan method13 showed a statistically significant difference.\n\nSD - standard deviation, IV - inverse variance, CI - confidence interval.\n\nThe pooled outcomes from 14 studies,1,7,21,25,27,30,32,34,37–40,42,44 with total sample size of 257 and 237 in the radiographic and POR group, respectively, the standardised mean difference (SMD) value for HCG angle using random effect model was 0.82 [0.34, 1.29] and showed a statistically significant difference (P=0.0008) between the radiographic and POR group [Tau2 =0.68, Chi2 =75.09, I2 = 83%] (Figure 4). After subgroup analysis was done using a random-effect model, it was shown that the CBCT method25 had a statistically significant difference favouring the radiography technique (SMD, 1.69; 95% CI: 0.84–2.54; P<0.0001). For OPG method,1,7,21,27,30,32,34,37–40,42 radiographic method showed a statistically significant difference as compared to POR method (SMD, 0.65; 95% CI: 0.11–1.20; P=0.02) with 83% heterogeneity. LC method1 showed a statistically significant difference between the radiographic and POR group (SMD, 1.99; 95% CI: 1.22–2.77; P<0.00001). TMJ tomogram method44 did not show a statistically significant difference between the radiographic and POR method (SMD, 0.59; 95% CI: 0.34–1.29; P=0.06) (Figure 4).\n\nSD - standard deviation, IV - inverse variance, CI - confidence interval.\n\nThe pooled outcomes from 14 studies,1,7,21,25,27,30,32,34,37–40,42,44 with total sample size of 257 and 237 in the radiographic and POR group, respectively, the standardized mean difference (SMD) value for HCG angle using random effect model was 0.66 [0.18, 1.15] and showed a statistically significant difference (P=0.007) between the radiographic and POR group [Tau2 =0.67, Chi2 =77.39, I2=83%] (Figure 5). After subgroup analysis was done using a random-effect model, it was discovered that for the CBCT technique,25 there was a statistically significant difference favouring the radiography method (SMD, 2.28; 95% CI: 1.33–3.23; P<0.00001). For OPG method,1,7,21,27,30,32,34,37–40,42 radiographic method showed a statistically significant difference as compared to POR method (SMD, 0.55; 95% CI: -0.02–1.13; P=0.06) with 85% heterogeneity. LC method1 did not show a statistically significant difference between the radiographic and POR group (SMD, 0.50; 95% CI: -0.13–1.13; P=0.12). TMJ tomogram method44 did not show a statistically significant difference between the radiographic and POR method (SMD, 0.59; 95% CI: -0.03–1.21; P=0.06) (Figure 5).\n\nSD - standard deviation, IV - inverse variance, CI - confidence interval.\n\nTable 4 presents the results of sensitivity analysis of the right and left HCG angle for the dentate and edentulous patients. Studies of fair quality1,7,9,13,14,19–24,26,27,29–34,36,38,40–44 or good quality8,11,25,28,35,37,39 were excluded from sensitivity analysis. After excluding these studies, the HCG angle values for the right and left sides of dentate patients, as well as the right side of edentulous patients, did not show a significant difference when comparing the radiographic and POR groups, with the exception of the left HCG angle values in edentulous patients, which showed an adverse change. A reanalysis using the fixed-effect model revealed that the results were not unfavourable. Excluding subgroups from a single research did not result in a substantial improvement in the SMD of HCG levels. Also, the inclusion of single radiographic technique of only CBCT and only OPG showed that the outcomes were not adverse whereas when inclusion of only LC was considered no statistically significant difference was found between the radiographic and protrusive occlusal record group for the HCG value of right and left side for dentate patients (Table 4).\n\nFigure 6 represents the funnel plot comparing horizontal condylar guidance angle between radiographic techniques and protrusive occlusal records for HCG angle values of right and left side for dentate and edentulous patients resemble a symmetrical (inverted) funnel indicating lack of publication bias.\n\nCT - Computed tomography, SE - Standard error, SMD- Standardized mean difference, TMJ - Temporo-mandibular joint.\n\n\nDiscussion\n\nThe route followed by the condyle with reference to the articular eminence when the mandible advances protrusively or laterally from centric relation is referred to as the condylar path.45 It is an essential regulating element since it impacts mandibular motions and is unique to each patient.19\n\nProsthodontics considers an equivalent of condylar guiding on an articulator to be an essential requirement.46 Articulators are utilised to imitate the patient's interocclusal positioning and certain mandibular motions. The sophistication and adaptability of the articulator determine the precision with which mandibular motions are reproduced. Using proper technique, the semi-adjustable articulator is capable of replicating specific points on the path of the condyle during a protrusive movement, when set with an interocclusal positional record.47\n\nBecause condylar inclination values obtained with various planes of reference cannot be compared,48 the plane of reference is an important element to consider. As a result, the radiological pictures give sagittal reconstructions of the skeletal components, and the contour of the articular eminence may be utilised to help in establishing the condylar guiding inclination of a semi-adjustable articulator.19\n\nAs a result, the goal of this study was to compare the condylar guidance values acquired by tracing radiography pictures to those produced by interocclusal protrusive recordings of dentulous and edentulous patients. The values of horizontal condylar inclination vary with age, gender, and ethnicity. This review included 33 studies published from 2011 to 2020 conducted in various countries which directly compared both the techniques. The age of the included dentate and edentulous patients ranged 18-43 years and 35–75 years, respectively. In completely edentulous patients, condylar paths are determined by the following factors: the bony fossae, tone of the muscles responsible for mandibular movements and their nerve controls, limitations imposed by the attached ligaments, shape and movements of the menisci while in dentate patients during protrusive movement, as the mandible moves forward there is an influence of the anterior guidance which affects the exact path of the condyle, hence separate analysis of dentate and edentulous patients was conducted.49 Hence, the results of this systematic review can be applicable to a varied population range and also in conditions as close as possible to those observed in daily clinical practice.\n\nThe overall results of the present systematic review and meta-analysis indicated that the HCG angle values of the right and left side showed a statistically significant difference favouring the radiographic method as compared to POR for dentate as well as edentulous patients. The sub-group analysis showed that for CBCT and OPG images the SMD of HCG angle values for dentate and edentulous patients on right side and left side showed a statistically significant difference as compared to the POR method except for edentulous patients the left side HCG angle values did not show a statistically significant difference between the two groups, whereas for LC images the SMD of HCG angle values of right and left side did not show a statistically significant difference between the two groups, except in edentulous patients right side HCG angle values showed a significant difference between the two groups.\n\nThe methodologies applied in the studies differed considerably. In the clinical method, the protrusive jaw relation is used to set the condylar elements of the articulator so they will reproduce inclinations, which are exact or nearer to the patient’s temporomandibular articulation. In the included studies interocclusal protrusive wax records, Lucia jig and gothic arch tracers have been used in setting the condylar guidance in semi-adjustable articulators. As the HCG changes with amount of protrusion, for studies where protrusive wax records were used the amount of protrusion was kept same for all the patients at 6 mm and the same protrusive records were used for programming the articulator so it is important to keep the distance of protrusion the same.20,50 Once the protrusive jaw relation is established, the majority of research employed a Hanua Wide-vue semi adjustable articulator, while a few studies used a whip mix semi adjustable articulator to measure horizontal condylar inclination. The reference plane is used to calculate horizontal condylar inclination. Hanua articulators generate more precise angles since they mount the cast in reference to the Frankfort horizontal plane, whereas whip mix employs the nasion-porion plane as a reference plane.35,51 POR technique for measuring SCGAs, regardless of the material used, is inconsistent, lacks precision and has lower levels of reproducibility because of significant differences between the instruments,31,33 deformation or compression of the records, cast tipping due to improper adaptation of casts, force applied by the operator on record,32 changes in values with the degree of protrusion, the amount of overjet and overbite.1,8,33 Also, semi-adjustable articulators are unable to reconstruct the condylar movements adequately because of their fixed inter-condylar distance and straight condylar pathway.33 Christensen et al. demonstrated that radiographically measured condylar angles yielded higher values than intraoral recording techniques. The rigid mechanical principles controlling the motions of an adjustable articulator appear to be inapplicable to man's dynamic mandibular locomotor system.19,52\n\nPanoramic radiography, lateral cephalogram and CBCT are now widely used in diagnoses. Significantly higher condylar guidance angle values in panoramic radiograph as compared to protrusive interocclusal record are reported for this review. The review's results might be supported for any of the following basis. First, the panoramic radiography technique often yields a larger value than the actual, as demonstrated by Gilboa et al46 in their study, in which they discovered the sagittal condylar inclination to be seven degrees more on average than its true anatomic contour in dry skulls. Second, when the occlusal rims are kept in a protruded mandibular position, they exert significant pressure on the mucosa of the denture basal seat, depressing the resilient oral mucosa and bringing the inter-ridge distance closer, resulting in a narrower triangular wedge shaped space between the posterior part of the occlusal rims, similar to the Christensen's space found in natural dentition and documented by protrusive interocclusal records, resulting in a lower value for HCG when positioned in semi-adjustable articulators.20,21 While the results obtained by lateral cephalogram were comparable to that of POR. However, the reference line used in all the included studies is the same, there were variations in the results of the included patients studies which may be because of variation in patients head positioning leading to parallax errors, the models of the panoramic machine, magnification differences, image distortions, overlapping of the mandibular notch, coronoid process, zygomatic arch around TMJ in an OPG and LC as well as the quantitative measurements can be affected by the different operator’s perceptions.7,14,21,23,30,34\n\nThe glenoid fossa and the AE can be easily identified since CBCT gives a three-dimensional information for both sides without superimpositions. For both dentate and edentulous individuals, the mean sagittal condylar values obtained from CBCT are slightly greater than those obtained from POR on both sides. Similar results were obtained by the individual studies included in the review, where in the study by Vadodaria43 condylar guidance obtained by CBCT were about 10° more than clinical methods were testified by Jerath et al,25 Kwon et al31 and Naqash et al33 where HCG angle values obtained from CBCT measurements being 5°–6° higher than those from protrusive occlusal records. The most significant advantage of CBCT is that it produces unique pictures that demonstrate characteristics in 3D that intraoral, panoramic, and cephalometric images do not. Cursor-driven measurement methods offer the physician an interactive real-time dimensional evaluation capability. Measurements taken on a computer screen are free of distortion and magnification. Furthermore, CBCT has other advantages such as superior image quality, employing a narrower field of view for a shorter check time, compatibility with various radiographic arrangements for image output, and ease of setup of minimum units in a general clinical context. These CBCT preferences may be utilised to determine the condylar position during dynamic registration in edentulous and dentulous patients and precisely locate the condyle.8,43,53 The main drawback of utilising CBCT is the expensive cost of the equipment.54 For, CT scan and TMJ tomogram radiographic technique only one study in each group was included, hence it is difficult to draw conclusions for these techniques.\n\nNonetheless, there are several limits to this evaluation. The clinical variability among the selected studies could not be completely avoided. The studies' sample sizes were limited, resulting in a lack of statistical power. None of the investigations correlated HCG angle measurements to actual MRI images, which are the gold standard in diagnosing TMJ problems.9 The eligible studies provided less evidence for inter and intra examiner reliability for radiographic scans and POR. Also, separate analysis was not performed for different methods of recording POR (protrusive wax records, jig’s method and gothic arc tracing), number of missing teeth for partially edentulous patients as well as comparison between different radiographic methods and right and left HCG angles was also not performed. However, to rule out potential causes of heterogeneity, subgroup and sensitivity analyses were done separately for dentate and edentulous individuals for the right and left HCG angle. Likewise, it was difficult to rule out the clinical heterogeneity occurring because of type of radiographic machine, tube voltage, selection of landmarks, head positioning and software capabilities. Only, seven studies were rated as good quality studies, exhibiting a low risk of bias suggesting that in future, high-quality of in-vivo studies assessing the reliability and accuracy of radiographic scans and POR with consistent outcome parameter should be conducted.\n\n\nConclusions\n\nThe present systematic review and meta-analysis concluded that for the dentate and edentulous patients, the right and left HCG angle values determined by radiographic method showed statistically significant difference as compared to the protrusive occlusal records. Yet, in clinical general practice, the approach most used to assess horizontal condylar inclination is by recording protrusive interocclusal records; however, if not managed properly, this method may result in restoration with distinctive errors. The numerous radiography approaches available through CBCT, OPG, and LC provide unique opportunities to minimise mistakes that may occur as a result of material mishandling while eliminating time-consuming procedures. Therefore, a clinically applicable HCG angle to program semi-adjustable dental articulators can be obtained by adjusting the value measured using CBCT images and pantographic tracings.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: PRISMA checklist for ‘Accuracy of radiographic and protrusive occlusal record methods in determining condylar guidance angles: a systematic review and meta-analysis’ https://doi.org/10.17605/OSF.IO/WAQNJ.55\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\nPaul R, Das S, Bhattacharyya J, et al.: A study on the accuracy of horizontal condylar guidance values in edentulous patients using preprosthetic diagnostic radiographs. The Journal of Indian Prosthodontic Society. 2018; 18: 263–270. 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Journal of Dental and Orofacial Research. 2017; 13: 35–42.\n\nGilboa I, Cardash H, Kaffe I, et al.: Condylar guidance: Correlation between articular morphology and panoramic radiographic images in dry human skulls. J. Prosthet. Dent. 2008; 99: 477–482. PubMed Abstract \| Publisher Full Text\n\nDonegan S, Christensen L: Sagittal condylar guidance as determined by protrusion records and wear facets of teeth. Int. J. Prosthodont. 1991; 4: 469–472.\n\ndos Santos J , Nelson S, Nowlin T: Comparison of condylar guidance setting obtained from a wax record versus an extraoral tracing: A pilot study. J. Prosthet. Dent. 2003; 89: 54–59. PubMed Abstract \| Publisher Full Text\n\nGajapathi B: A comparative study of condylar guidance of edentulous patients recorded from protrusive records with anterior teeth and gothic arch tracing –an in vivo study. Int. J. Sci. Res. 2019; 8: 1–3.\n\nPosselt U, Skytting B: Registration of the condyle path inclination: Variations using the Gysi technique. J. Prosthet. Dent. 1960; 10: 243–247. Publisher Full Text\n\nSantos J, Nelson J, Nummikoski P: Geometric Analysis of Occlusal Plane Orientation Using Simulated Ear-Rod Facebow Transfer. J. Prosthodont. 1996; 5: 172–181. PubMed Abstract \| Publisher Full Text\n\nChristensen L, Slabbert J: The concept of the sagittal condylar guidance: biological fact or fallacy?. J. Oral Rehabil. 1978; 5: 1–7. Publisher Full Text\n\nBarghan S, Tetradis S, Mallya S: Application of cone beam computed tomography for assessment of the temporomandibular joints. Aust. Dent. J. 2012; 57: 109–118. PubMed Abstract \| Publisher Full Text\n\nMuddugangadhar B, Mawani D, Das A, et al.: Comparative evaluation of condylar inclination in dentulous subjects as determined by two radiographic methods: Orthopantomograph and cone-beam computed tomography – An in vivo study. J. Indian Prosthodont. Soc. 2019; 19: 113–119. PubMed Abstract \| Publisher Full Text\n\nLuke AM, Kuriadom ST, George JM, et al.: Accuracy of radiographic and protrusive occlusal record methods in determining condylar guidance angles: a systematic review and meta-analysis.2021, December 10. Publisher Full Text" }	[ { "id": "121335", "date": "08 Mar 2022", "name": "Vineet Vinay", "expertise": [ "Reviewer Expertise Systematic Review and Meta-analysis", "Research design", "Statistics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe systematic review is conducted properly. The best features of this systematic review are the process of search strategy, data extraction, and quantitative synthesis of the results.\n\nThe language of the manuscript is up to the mark.\n\nKindly look for more systematic reviews in the same line and conduct Umbrella review in future.\n\nThe hard work of the researchers is clearly depicted in the conduct and writing of the manuscript.\n\nAs a statistician, I can say that the sensitivity and subgroup analysis is a correctly done and it fulfills the requirements of the research.\n\nHowever I personally believe that any research cannot be perfectly done. In this manuscript, there are limitations regarding the number of studies included, however, I believe that it's not in the hand of the researchers.\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": "7958", "date": "16 Mar 2022", "name": "Dian Agustin Wahjuningrum", "role": "Author Response", "response": "Thank you so much, respected reviewer, for the positive comments to our attempt to perform a systematic review and meta analysis. We will surely look for more systematic reviews in the same context and conduct Umbrella review in future. Appreciation from a person who expertise in statistic, we are highly obliged. Thank you once again, respected reviewer." } ] } ]	1	https://f1000research.com/articles/11-105
https://f1000research.com/articles/11-104/v1	27 Jan 22	{ "type": "Method Article", "title": "A population of in silico models identifies the interplay between Nav 1.8 conductance and potassium currents as key in regulating human dorsal root ganglion neuron excitability", "authors": [ "Oliver J. Britton", "Blanca Rodriguez" ], "abstract": "Background: The Nav 1.8 sodium channel has a key role in generating repetitive action potentials in nociceptive human dorsal root ganglion neurons. Nav 1.8 is differentiated from other voltage-gated sodium channels by its unusually slow inactivation kinetics and depolarised voltage-dependence of activation. These features are particularly pronounced in the human Nav 1.8 channel and allow the channel to remain active during repolarisation. Gain-of-function mutations in Nav 1.8 have been linked to neuropathic pain and selective blockers of Nav 1.8 have been developed as potential new analgesics. However, it is not well understood how modulating the Nav 1.8 conductance alters neuronal excitability and how this depends on the balance of other ion channels expressed by nociceptive neurons. Methods: To investigate this, we developed a novel computational model of the human dorsal root ganglion neuron and used it to construct a population of models that mimicked inter-neuronal heterogeneity in ionic conductances and action potential morphology Results: By simulating changes to the Nav 1.8 conductance in the population of models, we found that moderately increasing the Nav 1.8 conductance led to increased firing rate, as expected, but increasing Nav 1.8 conductance beyond an inflection point caused firing rate to decrease. We found that the delayed rectifier and M-type potassium conductances were also critical for determining neuronal excitability. In particular, altering the delayed rectifier potassium conductance shifted the position of the Nav 1.8 inflection point and therefore the relationship between Nav 1.8 conductance and firing rate. Conclusions: Our results suggest that the effects of modulating Nav 1.8 in a nociceptive neuron can depend significantly on other conductances, particularly potassium conductances.", "keywords": [ "DRG neurons", "hDRG neurons", "pain", "Nav 1.8", "computational modelling", "population of models" ], "content": "\n\n\n\nScientific benefits\n\n\n\n• Software and model allow simulation of human dorsal root ganglion (hDRG) neuron electrophysiology incorporating human-specific data at action potential (AP) and ion channel levels where possible.\n\n• Population of models incorporates observed experimental variability in hDRG neuron AP biomarkers and mimics variability in ion channel densities, allowing the effects of this inter-neuronal variability to be simulated.\n\n• Use of computational modelling allows large numbers of experimental conditions to be simulated rapidly and for voltage traces and individual ionic currents in each simulation to be simultaneously recorded and analysed.\n\n3Rs benefits\n\n\n\n• These simulations have the potential to replace experiments using animal DRG neurons, particularly experiments for hypothesis generation and for studying electrophysiological mechanisms.\n\n• These simulations can also be used alongside existing experimental approaches early in drug development to refine target identification and candidate selection, further reducing the need for animal studies.\n\n• The current model can be iterated on and improved as new human-specific data becomes available, increasing the accuracy of the model and the range of animal studies it could potentially replace.\n\nPractical benefits\n\n\n\n• Many simulations can be run in a short amount of time, allowing rapid investigation of many different simulated experimental conditions.\n\n• The models, population of models approach and simulation runner are implemented within a Python software package that is freely available and open source. Python itself is widely used by the scientific research community.\n\nCurrent applications\n\n\n\n• Studying hDRG neuron AP electrophysiology and the roles of different ionic currents in these neurons incorporating the effects of inter-neuronal variability in ion channel conductances.\n\n• Simulating the effects of ion channel block and enhancement, and of ion channel mutations, in hDRG neurons.\n\nPotential applications\n\n\n\n• Studying propagation of action potentials in an extended model including the full hDRG neuron geometry.\n\n\nIntroduction\n\nThe sodium channel Nav 1.8 is selectively expressed in small diameter dorsal root ganglion (DRG) neurons and has an important and unique role in pain signalling, due to its slow and depolarised kinetics of inactivation [Akopian et al., 1996; Sangameswaran et al., 1996] and fast recovery from inactivation [Dib-Hajj et al., 1997] compared with other voltage-gated sodium channels. Human Nav 1.8 has particularly slow inactivation and larger persistent current compared to rodent Nav 1.8 [Han et al., 2015]. Gain-of-function mutations in Nav 1.8 contribute to painful peripheral neuropathy [Faber et al., 2012; Huang et al., 2013; Han et al., 2014], and Nav 1.8 has been shown to support repetitive firing and the characteristic broad shoulder of DRG neuron action potentials [Renganathan et al., 2001; Blair and Bean, 2002]. Nav 1.8 has also been identified as a potential therapeutic target, due to its effects on DRG neuron excitability, and selective blockers of the channel have been developed [Jarvis et al., 2007; Payne et al., 2015]. However, to date, there has only been one clinical trial of a selective Nav 1.8 blocker, which was an unsuccessful trial of PF-04531083 [Payne et al., 2015] for post-surgical dental pain.\n\nIt is therefore important to better understand how Nav 1.8 affects the excitability of human DRG (hDRG) neurons. However, the effect of an ion channel on neuronal electrophysiology depends not only on its individual kinetics and expression level but also on the background ensemble of other ion channels expressed in the neuron. For example, in DRG neurons expressing Nav 1.8, a gain-of-function Nav 1.7 mutation caused hyper-excitability while in sympathetic ganglion neurons without Nav 1.8 the same mutation causes hypo-excitability [Rush et al., 2006]. Understanding how variation in multiple ionic currents will interact with each other, not just on a qualitative level (whether a channel is expressed or not) but on a quantitative level (the level of expression of each channel), is challenging. This is an important consideration though, as ion channel densities have been shown to have high inter-neuronal variability [Schulz et al., 2006]. They can cause different neurons to respond differently to the same experimental conditions, e.g. application of the same concentration of a drug. Pathological conditions also substantially alter expression of multiple channels in DRG neurons [Black et al., 2004; Cao et al., 2010; Zhao et al., 2013].\n\nIn this study, we investigate the effects of modulating the Nav 1.8 conductance (GNav 1.8) on hDRG neuron excitability using computational modelling and simulation. We hypothesised that altering the Nav 1.8 conductance would be positively correlated with firing rate, but that the quantitative change in firing rate would depend on the balance of other conductances in a particular model. To test this hypothesis, we develop a novel model of the hDRG neuron based on human-specific ionic currents. We construct a population of 328 experimentally calibrated human DRG neuron models [Britton et al., 2013; Marder and Taylor, 2011; Prinz et al., 2003/4] that integrate data from electrophysiological recordings of human DRG neurons at the cellular [Davidson et al., 2014] and ion channel levels [Han et al., 2015; Vasylyev et al., 2014; Huang et al., 2014]. Each model in the population shares the same underlying kinetics and equations for the eight ionic currents in the model, but has a different set of ionic conductances, reflecting inter-neuronal heterogeneity in ion channel densities. Importantly, every model in the population produces action potential behaviour in range with the experimental variability reported in human DRG neurons by Davidson et al. [2014] when simulated with equivalent protocols and measured with eight action potential parameters.\n\nThis study advances the replacement of animal DRG neurons for research into human DRG neuron electrophysiology by developing the first computational model of human DRG neuron electrophysiology and providing open-source software to run simulations using the model. This software allows interested users to begin using the model with the population of models approach described in this study for modelling inter-neuronal variability. The main situations where we think our model would be particularly suited to replacing the animal DRG neuron experiments are for hypothesis generation, as many different experimental conditions can be rapidly simulated and analysed; and for detailed analysis of ionic current behaviours as each current can be output and visualised.\n\n\nMethods\n\nIn this study, we use a non-standard modelling approach based on experimentally calibrated populations of models [Britton et al., 2013; Marder and Taylor, 2011]. This methodology, which originated in neuroscience [Prinz et al., 2003, 2004; Marder and Taylor, 2011], has also been used extensively to model the electrophysiology of cardiac cells [Britton et al., 2013; Passini et al., 2017], but to our knowledge this is the first time it has been used to study DRG neurons. This approach allows us to integrate the observed experimental variability in hDRG neuron action potential (AP) parameters [Davidson et al. 2014] into the modelling process, instead of the standard approach where a single model parameterised to reflect a typical neuron is used [Marder and Taylor, 2011].\n\nHere we explain the population of models methodology. For more detail, see [Britton et al., 2013; Marder and Taylor, 2011; Muszkiewicz et al., 2016]. The main idea is to create a group of neuronal models that all have different underlying conductance values (representing inter-neuronal variability in ion channel densities) that, when simulated, all generate voltage traces with AP biomarkers that are within the range of values observed experimentally in hDRG neurons.\n\nTo create our population of models, we first construct a baseline model. This model is a Hodgkin–Huxley-type neuronal AP model [Hodgkin and Huxley, 1952] comprising a set of equations describing the changes in membrane voltage and ionic current gating variables over time. We then select parameters in the model to be varied to represent inter-neuronal variability. In this study we make the assumption that ion channel expression varies from neuron to neuron but that the structure of each ion channel type does not. Therefore, we randomly vary all conductances in the baseline model but leave the kinetics parameters of each ionic current constant. Through random variation of all conductances we obtain a pool of candidate models, each with a different set of conductances. We simulate each model to find its rheobase, record its voltage trace and from this trace calculate AP biomarkers. We then calibrate the population by comparing each AP biomarker in each model to the experimental range for that parameter. If any AP biomarker for a model is outside the experimental range, the model is rejected. If all tested AP biomarkers are within their experimental ranges then the model is accepted. The final population of models consists of only the accepted models. All models in the population have a different set of conductances but each model is in range with experimental data on AP parameters of ex vivo hDRG neurons.\n\nFor this study, we chose to model the human dorsal root ganglion neuron soma, rather than the axon or full DRG neuron geometry. This is because the soma was the section of the neuron that was patched and recorded from in both the experimental data we used to calibrate the population [Davidson et al., 2014] and in the vast majority of DRG neuron electrophysiological studies, due to its large size compared with the axon. Our aim was to create a model that could be directly compared against results from the experimental system from which data are available.\n\nWe developed a baseline model, which was a single model with no variability in ionic conductances. The baseline hDRG model contains eight ionic current models: INav 1.7 [Vasylyev et al., 2014]; INav 1.8 [Han et al., 2015]; INav 1.9 [Huang et al., 2014]; IKdr [Sheets et al., 2007]; an A-type potassium current (IKA) [Sheets et al., 2007]; an M-type potassium current (IKM) [Maingret et al., 2008]; a leak potassium current (IKleak); and a hyperpolarization-activated current (Ih) [Kouranova et al., 2008]. All current models used a Hodgkin–Huxley formulation.\n\nThe kinetics (voltage dependencies of activation and inactivation, and time constants) of each ionic current were not varied in this study, while all eight conductances were simultaneously varied. These choices are based on the assumptions that: 1) kinetic parameters depend primarily on ion channel structure, which as a simplifying assumption for modelling we assume will not vary between different DRG neurons; 2) conductances represent the density of each ion channel type in the cell membrane, and these vary substantially between neurons [Schulz et al., 2006].\n\nThe soma was modelled as a single cylindrical compartment with length=30 μm, diameter=46 μm, and cytoplasmic resistivity=1 Ω/cm following the values used in the DRG modelling study by Choi and Waxman [Choi and Waxman, 2011]. A cylindrical geometry was used in line with how the NEURON modelling software represents all neuronal compartments. The specific membrane capacitance was set to the commonly used value of 1 μF/cm2. The internal and external ionic concentrations were fixed to match the experimental conditions used by Davidson et al. [2014] with extracellular [Na+]=145 mM, intracellular [Na+]=5 mM, extracellular [K+]=3 mM, and intracellular [K+]=135 mM.\n\nThe equations for the baseline model can be found in the supplementary material and source code is provided in the data supplement.\n\nWe used data on experimental AP biomarkers of ex vivo human DRG neurons from Davidson et al. [2014] to calibrate the population of models against experimental data. This dataset included data from recordings of 141 ex vivo human DRG neurons from five organ donors.\n\nEach neuron’s soma was recorded under two stimulus protocols, an 800-ms step stimulus and a 500-ms ramp stimulus. In both cases, stimulus current was increased in steps of 50 to 100 pA until AP threshold was reached for that neuron, and AP biomarkers were calculated from traces at that stimulus current amplitude.\n\nMeasuring AP properties\n\nWe used eight AP biomarkers to quantify the electrophysiological properties of hDRG neuron APs: AP threshold voltage; AP peak voltage; AP slope maximum; AP slope minimum; AP full width; after-hyperpolarisation time constant; resting membrane potential; and rheobase. Each biomarker was calculated as follows:\n\nRheobase\n\nRheobase was calculated for each model by running step current simulations with increasing stimulus current amplitudes in increments of 100 pA from 0 pA to up to 5000 pA until the first simulation in which one or more action potentials were detected. The stimulus amplitude of that simulation is the rheobase for that model.\n\nAP threshold voltage\n\nFollowing the approach used by Davidson et al. [2014], AP threshold voltage was calculated from a ramp current simulation and defined as the voltage during the upstroke of an action potential at which the voltage-time gradient first surpasses 5 mV/ms.\n\nAP peak voltage\n\nAP peak voltage was calculated as the peak voltage attained during an action potential.\n\nAP slope maximum and minimum\n\nAP slope minimum and maximum was calculated as the maximum voltage–time gradient and minimum voltage–time gradient during an action potential.\n\nAP full width\n\nAP full width for an action potential was calculated as the duration between the time at which the voltage first crossed the AP threshold voltage from below, and the time at which the voltage first crossed the AP threshold voltage from above.\n\nAfter-hyperpolarisation time constant\n\nThe time constant (τ) of the after-hyperpolarisation of an action potential was calculated by fitting the after-hyperpolarisation to the following single-exponential model with a curve fitting algorithm and extracting the value of τ:\n\nThe after-hyperpolarisation of an action potential was defined as the period from the minimum voltage that occurred after the AP peak, up to the point at which the voltage gradient exceeded 5 mV/ms or 50 ms had elapsed, whichever came first.\n\nResting membrane potential\n\nThe resting membrane potential of a model was defined as the minimum voltage that occurred during the last 10% of a simulation in which no stimulus current was applied.\n\nTo create the initial pool of candidate models, all conductances in the baseline model were simultaneously and randomly sampled in a range of 0–2-times the baseline model value for that conductance (Extended data - Equations_and_conductance_densities.docx – Conductance densities table) to create 20,000 candidate models with the same ionic currents and equations but different conductance values. Conductance sampling was performed using Latin hypercube sampling (McKay et al. 1979), which is a technique that evenly samples each conductance without exhaustively sampling all possible conductance combinations. We used Latin hypercube sampling as sampling all possible combinations of eight conductances and simulating the resulting models is not computationally feasible, even if each conductance is sampled coarsely. The range of 0–2-times baseline was chosen to allow a broad range of different conductance profiles within the population. Previous studies of neurons have shown high variability in mRNA expression and current densities between healthy neurons of the same type [Schulz et al., 2006]. We set the minimum of our sampling range to zero (equivalent to no expression of functional ion channels) to allow our population to potentially include model neurons that produce a normal AP under normal conditions even without substantial expression of all ionic currents in the model. This can represent conditions such as a loss of function mutation that may not affect normal neuronal function but could change the response when conditions are changed, e.g. if another channel is blocked by a drug.\n\nEach model was simulated under 800-ms step and 500-ms ramp protocols at its individual rheobase to match the stimulation protocols used in Davidson et al. [2014]. From the voltage traces from these simulations we extracted values for the eight AP biomarkers described previously: AP threshold voltage; AP peak voltage; AP slope maximum; AP slope minimum; AP full width; after-hyperpolarisation time constant; resting membrane potential; and rheobase.\n\nWe then calibrated our candidate models against experimental ranges of these AP biomarkers calculated from data in Table 3 of Davidson et al. [2014]. The experimental range for each AP biomarker was defined as the range spanned by the mean ±1.5 standard deviations of the data for that parameter. The choice of 1.5 standard deviations was partly arbitrary but was chosen so that the range would include approximately 90% of the total probability density of the distribution of each AP biomarker assuming a normal distribution. This was so the population would capture a large proportion of total variability in each AP biomarker while excluding extreme outliers.\n\nIn this study, we performed simulations to investigate the effects of altering the GNav 1.8 conductance, as well as the GKdr and GKM conductances, across the whole population. In each of these simulations, we implemented this by multiplying the conductance of each model in the population by a fixed scaling factor. This mimics how drug block would affect the conductances of different neurons. For example, two neurons with GNav 1.8 values of 1 and 2 respectively subject to a scaling factor of 0.5 would have their GNav 1.8 values reduced to 0.5 and 1, respectively, equivalent to 50% Nav 1.8 channel block.\n\nAll simulations were run using NEURON version 7.5 (RRID:SCR_005393) [Carnevale, 2006], through NEURON’s Python (RRID:SCR_008394) interface. NEURON uses the adaptive time step ODE solver CVODE [Hindmarsh et al., 2005] to solve model equations. All simulations were recorded at 40 kHZ (NEURON’s default) then down-sampled to 20 kHz to match the sampling rate used by Davidson et al. [2014]. All analysis and visualisation was performed using Python 3.7. The Python module 'drgpom' we developed to run NEURON simulations on populations of models and analyse the results is included in the extended data and the most up to date version can also be found at [https://github.com/oliverbritton/drg-pom]. The module is also available on the Python online package repository Pypi [https://pypi.org/] and so can be downloaded and installed with a single command. The module comes with a series of examples to guide users in how to use the module, which we hope will help potential users, who have not used populations of models before, run their own simulations.\n\nAn advantage of computer simulations is the ability to simultaneously record the voltage trace and all individual ionic currents in each simulation. To visualise the relative contributions of all ionic currents throughout simulations we used a visualization technique called a currentscape [Alonso and Marder, 2019]. A currentscape shows the voltage trace and the fractional contribution of each ionic current throughout a current clamp simulation. Each currentscape shows the voltage trace from a simulation at the top, followed by plots showing the fractions of each outward current in the model and each inward current in the model. This allows us to see which currents are influential at each phase of the action potential, and how this differs from model to model.\n\nThis guide for installing the drgpom package for Python assumes you are installing on a Windows machine and has been tested on Windows 7 and Windows 10, and should also work on Linux (drgpom has been tested on the Fedora Linux distribution).\n\nThe use of this software requires a basic familiarity with Python. One of the best places for scientists to learn Python for scientific computing is to follow the lessons from the Software Carpentry website: https://software-carpentry.org/. It also requires installation of Python 3 and the NEURON simulation software, both of which are freely available and open source.\n\n\n\n1. Download and install the Anaconda Python 3.7 distribution (https://www.anaconda.com/). This is a Python distribution for scientific computing including most of the key libraries needed for drgpom.\n\n2. Install NEURON (https://neuron.yale.edu/neuron/). NEURON is simulation software that is widely used in computational neuroscience for simulating neuronal electrophysiology. We use it to run the individual simulations and implement the models in drgpom.\n\nInstalling drgpom and compiling models\n\nThe simplest way to install drgpom is to download it using pip, which is a Python tool for automatically download and installing packages from online repositories. If you have installed Anaconda command window, pip will come installed with it.\n\nYou can install drgpom using pip by opening a command window (in Windows this will be a command prompt and can be opened by running the command cmd from the Run option in the Start Menu, in Linux this will be a terminal). From there run the command:\n\nAlternatively, download the latest version from the github repository at: https://github.com/oliverbritton/drg-pom/, or the version used for this study from the repository linked in the data availability statement. To install, first unpack the download, then navigate to its directory, open a command window and run the command:\n\nNow that drgpom is installed, the ionic current model files need to be compiled by NEURON before being used. To do this in Windows, follow these instructions:\n\nhttps://www.neuron.yale.edu/neuron/static/docs/nmodl/mswin.html\n\nFor Linux follow these instructions:\n\nhttps://www.neuron.yale.edu/neuron/static/docs/nmodl/unix.html\n\nIn either case, the model files that need to be compiled are located in the models directory in your installed drgpom folder. If you have installed Anaconda, these will be located in your Anaconda install directory within the Lib/site-packages/drgpom/models subfolder.\n\nUsing drgpom to run and analyse simulations\n\nIncluded in drgpom's examples folder is a Jupyter notebook, showing various examples of how to run simulations and save, load and view results using drgpom. Jupyter notebook (https://jupyter.org/) is a tool that allows code, data and visualisations to be integrated together in a single interactive environment that runs in a web browser.\n\nIf you have installed Anaconda you can start Jupyter notebook either by opening a command window and typing:\n\nor alternatively in Windows you can open the Anaconda folder in your start menu and click the Jupyter notebook icon in there. This will open the notebook in your default web browser, from there, navigate to the notebook of examples (examples_notebook.ipynb) in the examples folder of your drgpom installation and click to open it. A Jupyter notebook is composed of multiple cells containing either text or code. Each cells containing code can be run by selecting it and clicking the run button at the top of the screen or pressing Ctrl + Enter. All code and text in a notebook can be edited so we recommend making a copy of the notebook to experiment with.\n\nRunning an example simulation\n\nTo run an example simulation, open a command window in your drgpom directory (if using Anaconda this will be in the Lib/site-packages/drgpom/examples folder in your Anaconda directory) and run the script example_simulation.py in Python by entering the command:\n\nThis should produce some output including the message “This simulation has finished running” and the results of the simulation along with simulation metadata such as model details and parameter values will be saved in a file called “example_population.pkl”. This is a pickle file, which is a format for saving Python objects so they can be reloaded. A set of images will also be saved showing the voltage traces from each model in each simulation. To see how to view results from this simulation see the example notebook.\n\nConfiguring your own simulations using a pre-existing population of models\n\n1. To run a new simulation using the provided population of models, copy “example_simulation.py” and alter the options parameters to configure your simulation. The simulation parameters for this type of simulation are described in Table 1 below.\n\n2. To run your simulation, open a command window or Jupyter notebook in the directory the simulation script is saved in.\n\n3. If using a command window type: python script_name.py cores=n to run the script, where n is the number of CPU cores you want to use (or don't include cores=n to use all but one core by default). If using Jupyter notebook, run the code %run script_name.py cores=n. In both cases the simulation will run, leave the notebook or prompt running until it has finished. When the simulation has finished you will see a message that looks like:\n\n“Time taken on 4 cores = 1234 seconds. Current population saved to example_population.pkl. This simulation has finished running.”\n\n\n\n4. You can now open your population results by following the examples in the example Jupyter notebook. To open the example notebook follow the instructions in the “Using drgpom to run and analyse simulations” section above.\n\nCreating and calibrating a new population of models\n\n1. To create and calibrate a new population of models use example_population_creation.py as a template and alter the parameters in Table 1 above and Table 2 below as required. Simulation parameters are the same as those given in Table 1 for using an existing population of models.\n\n2. Once you have created your simulation file, open a command window, navigate to the directory the file is in and enter:\n\n\n\nwhere your_simulation.py is the name of your simulation file.\n\n3. The simulation will now run, do not close the window before it has finished as this will halt the simulation.\n\n4. Once the simulation is completed the results will be saved to a file that can be opened in Python. The examples notebook provides example code showing how to view your results.\n\nNotes on the model_details parameter\n\nThe model_details parameter defines the ion channel models that will be included within each model in the population, and which parameter(s) in each ion channel model will be varied. To define this parameter we first initialise a dictionary called model_details containing another dictionary called mechanisms that will store the details of each ion channel model:\n\nEach ionic current model to be included in the model, and each parameter to be varied within that ion channel model is defined as follows:\n\n‘nrn_mech_name’ is the name of the ion channel model in NEURON. To find this name open the.mod file for the relevant current model in the Models directory of the repository and look for the line beginning SUFFIX. This gives the name of the mechanism in NEURON.\n\n‘param_name’ is the name drg-pom will know that parameter by, for example ‘GNav 1.7’. You can choose this parameter name to be whatever you want, ideally something clear and memorable. These parameter names should correspond to those used in parameter_data to define the scaling ranges each parameter will be sampled over.\n\n‘param_name_in_nrn’ is the name that parameter is known by in NEURON. The format of this name is the name of that parameter in the.mod file of its associated ionic current model, followed by an underscore and nrn_mech_name. For example conductances are generally given the variable name ‘gbar’, so the conductance for the kleak ion channel model is named ‘gbar_kleak’. See the example_population_creation.py file for an example of a complete model_details parameter.\n\nRunning a simulation with multiple channel block conditions on multiple channels\n\nThe simulation script vary_amp_and_gnav18.py in the examples directory provides an example of a simulation design similar to those used in the paper where multiple simulations are run simulating changes in multiple conductances at multiple different levels. This example can be used as a template to design your own simulation studies.\n\n\nResults\n\nThe population of hDRG neuron AP models consisted of 328 models each of which had the same underlying equations but a different combination of eight ionic conductances. All models in the population had substantially different combinations of conductances, but all of them produced AP biomarkers at rheobase that were within 1.5 standard deviations of the mean values of each parameter, based on data recorded from ex vivo hDRG neurons [Davidson et al., 2014].\n\nVoltages traces from the population of models (Figure 1A) show they reproduce key characteristics of the hDRG neuron AP, including its characteristic broad shoulder [Davidson et al. 2014, Han et al., 2015], the slow rise to threshold followed by a rapid upstroke, and the presence of an after-hyperpolarization following repolarization. A schematic of the baseline model is shown in Figure 1B.\n\nA) Overlaid voltage traces from every model in the population stimulated with a step stimulus at each model’s rheobase. Each model was calibrated against hDRG neuron AP biomarker ranges calculated from data in Davidson et al. [2014].\n\nB) Model schematic. The model approximates the hDRG neuron soma as a single compartment Hodgkin-Huxley model with 8 ionic currents. Arrows indicate the usual direction of current flow for each current. Each model in the population has the same 8 ionic currents but different channel densities of each to simulate inter-neuronal variability in ionic conductances.\n\nThe traces in Figure 1A were produced by models with a diverse range of conductance profiles (Figure 2). Most conductances (except for GNav 1.8 and GKA) span the sampled conductance range. For GNav 1.8 and GKA, the accepted models only have a subset of the sampled conductance range (GNav 1.8: 0.15 – 1.04, GKA: 0 – 1.22). For GNav 1.8, this is because models with low GNav 1.8 have peak voltages that are below the experimental range, and models with high GNav 1.8 have threshold voltages that are below the experimental range. For GKA, no model with high GKA had a combination of threshold voltage, peak voltage, and after-hyperpolarisation time constant that was simultaneously within the experimental ranges of these three AP biomarkers.\n\nHistograms on the diagonal panels show the distributions of individual conductances. Scatter plots show distributions for all pairs of conductances. Conductances are given in terms of a dimensionless scaling factor applied to the baseline conductance values (which are provided in the supplementary material).\n\nMost of the pairs of conductances are uncorrelated (Figure 2), however GNav 1.8 and GKdr have a strong positive correlation (r = 0.82). This correlation is caused by calibrating on a combination of three biomarkers: threshold voltage, peak voltage and AP full width parameters, as removing all of these AP biomarkers from the calibration process also removes the correlation between GNav 1.8 and GKdr and removing any one of them reduces the correlation coefficient. This indicates that in our model the balance of these two currents is important throughout the action potential, from upstroke (threshold, peak voltage) to repolarisation (AP full width).\n\nAs can be seen in Figure 2, we found many different combinations of conductances that produced viable models consistent with observed experimental variability in hDRG AP biomarkers. However, while each of these models produced similar voltage traces, the different conductances in each model mean that the balance of underlying ionic currents in each model were substantially different. Figure 3 shows the conductances and relative magnitudes of each current in six models from the population using a visualisation technique called a currentscape [Alonso and Marder, 2019] (see Methods for details).\n\nA: Conductance scaling factors for six models selected to show a wide variety of different conductances and ionic current profiles.\n\nB-G: Currentscapes [Alonso and Marder, 2019] showing the fractions of each ionic current active during a simulated step stimulus. Each plot shows, from top to bottom, the voltage trace, the total outward current, the fraction each outward current contributes to the total outward current during the simulation, the fraction each inward current contributes to the total inward current during the simulation, and the total inward current.\n\nWe see that during quiescence the primary active currents are IKdr (purple) and IKleak (pink) for the outward currents and Ih (brown) and INav 1.9 (green) for the inward currents. The relative proportions of these currents are however highly variable from model to model. Similarly, the main currents during the upstroke are INav 1.7 (red) and INav 1.8 (blue), but some models have a large contribution by Nav 1.7 and in some models the contribution is very small, while the contribution of INav 1.8 is uniformly large.\n\nDuring the body of the AP, in all models IKdr (purple) is the dominant outward current and INav 1.8 (blue) is the dominant inward current. These two currents appear to have unique roles in shaping the AP that cannot be performed by any of the other currents in our model. This can explain why GKdr and GNav 1.8 are highly correlated in Figure 2; the values of both conductances must be balanced against one another as no other currents can perform their roles in shaping the AP.\n\nFollowing repolarisation, the potassium currents IKA (orange) and IKM (yellow) are both important for shaping the after-hyperpolarization. However, the balance of these two currents differs from model to model. For example, the model in Figure 3E has most of its post-repolarisation outward current conducted through IKA with a very small contribution from IKM, while the model in Figure 3F relies primarily on IKM with a small contribution by IKA. As we show later, the balance of these currents is not as critical as the balance of GNav 1.8 and GKdr at rheobase, where only a single AP is fired. However, at higher stimulus amplitudes that support repetitive firing, the value of GKM becomes important for modulating firing rate.\n\nFollowing the evaluation of the population of hDRG neuron models shown in the previous section, we investigated how changing the Nav 1.8 conductance affects hDRG neuron excitability across a wide range of different backgrounds of other ionic conductances. We therefore simulated different levels of block and enhancement of the Nav 1.8 conductance in every model in the population, across a range of stimulus amplitudes. Figure 4A shows the GNav 1.8 scaling factor and stimulus amplitude parameters for the 441 simulations that we ran. For each simulation the 328 models in the population were each simulated under an 800 ms step stimulus current protocol. The Nav 1.8 conductance was varied by multiplying GNav 1.8 in each model by a scaling factor, which is similar to how drug block or enhancement would affect the conductance.\n\nA) Simulation study design showing the grid search design used over stimulus amplitude and GNav1.8. Each dot represents a simulation on all 328 models in the population with the given Nav 1.8 conductance scaling factor applied to all models, and an 800 ms step stimulus current applied with the given amplitude.\n\nB) Mean firing rate of the population for each of the different simulation conditions. Each square represents a simulation of all models in the population with a different pair of values for GNav 1.8 scaling factor and stimulus amplitude.\n\nC) Mean AP half width of the population for each of the different simulation conditions.\n\nFigure 4B shows the mean firing rate (APs/s) of the population of models in each simulation. Mean firing rate is not maximised at the highest GNav 1.8 we simulated but is instead maximised when GNav 1.8 is slightly above the baseline value (between 1.1 and 1.3 times baseline, depending on the stimulus amplitude of the simulation). If GNav 1.8 is increased beyond this point, the mean firing rate decreases. This result implies that increased expression of Nav 1.8 could lead to a decrease in firing rate. We also find that reducing GNav 1.8 to 0.5 times baseline or less is sufficient to stop repetitive firing at all of the stimulus amplitudes we simulated, indicating the importance of Nav 1.8 in our model for enabling repetitive firing.\n\nWe hypothesised that one reason for the decrease in firing rate as GNav 1.8 is increased could be increased AP width due to increased Nav 1.8 current during AP repolarisation. A wider AP could reduce the maximum firing rate of a neuron by lengthening the minimum time between subsequent APs. Unlike other sodium channels Nav 1.8 is active during AP repolarisation, contributes to the characteristic shoulder of the DRG action potential [Han et al., 2015], and is therefore important for determining AP duration. Figure 4C shows that mean AP half width (defined as the period during an AP in which voltage is continuously above a threshold halfway between the threshold voltage and peak voltage) increases steadily with increased GNav 1.8 and is insensitive to stimulus amplitude. For example, averaging over different stimulus amplitudes, mean half-width of the population changed from 3.6 ms at baseline (GNav 1.8 = 1) to 4.5 ms when GNav 1.8 is doubled (GNav 1.8 = 2).\n\nPlotting the relationship between half width and firing rate in individual models (Figure 5) shows that firing rates within the physiological range (approximately 0 – 25 AP/s) can occur across a range of half-widths, but the very high firing rates that some models in the population exhibit (up to approximately 120 AP/s) only occur at the lower half-widths found predominantly in simulations with lower GNav 1.8 scaling values (e.g. Figure 5, second panel from the top). However, at the lowest values of GNav 1.8, there is very little repetitive firing (Figure 5, top panel). Therefore, one of the effects of Nav 1.8 on hDRG neuron electrophysiology may be to support repetitive firing but also limit the maximum firing rate by broadening the AP.\n\nBlue dots show results from individual models in different simulations from the simulation design shown in Figure 4A. Red lines show mean values of plotted results. Descending panels show results from simulations with progressively larger GNav 1.8 scaling factors.\n\nWe noticed that a substantial fraction of models fired at a very high rate (> 20 AP/s) (Figure 5. This is above the physiological values reported from real hDRG neurons [Meyer and Campbell, 1981] but these models are still relevant because they exhibit a normal hDRG-like AP at rheobase. The ways that these models’ conductance profiles differ from models in the population that do not fire at a very high rate could indicate which currents act as brakes or enhancers of excitability when the Nav 1.8 conductance is altered.\n\nTherefore we divided the population of models into two groups based on the number of simulations from Figure 4A in which a model fired at a rate > 20 AP/s. The top 25% of models were assigned to the “most often rapidly firing” group” and the other 75% of models to the “all other models” group. Figure 6A shows the distribution of each conductance in each group. Interestingly, GKdr and GKM differ substantially between the two groups. GKM is much smaller in the most often rapidly firing group (0.64 ± 0.32) than the other models group (1.41 ± 0.43). GKdr is larger in the rapidly firing group (1.60 ± 0.44) compared to the other models group (1.28 ± 0.43). That GKdr is actually larger in the rapidly firing group suggests it is not just a lack of total potassium current conductance, and a corresponding lack of outward current to oppose repetitive firing, that causes these models to fire so rapidly.\n\nA) Conductance distributions of two subpopulations in the population of models. Blue histograms show conductance distribution for the top 25% of models ranked by the number of simulations in Figure 4 in which they fired at a very rapid rate (> 20 AP/s). Red histograms show the other 75% of models. Histograms of the two subpopulations are normalised as the number of models in each subpopulation were not equal.\n\nB) Results of varying GNav 1.8, stimulus amplitude and one of GKdr (top row) or GKM (bottom row). Each square in a plot shows the mean firing rate across the population of models for one simulation.\n\nWe therefore simulated the effects of modulating each combination of Nav 1.8 and one of the other seven conductances in the model to confirm whether the firing rate of models in the population were sensitive to GKdr and GKM, but no other conductances. Modulating either GKdr or GKM substantially altered the effects of varying GNav 1.8 on firing rate (Figure 6B), while the other five conductances showed little effect on firing rate (Figure 7). Modulating GKdr (Figure 6B, top row) altered firing frequency by increasing the Nav 1.8 values required for high frequency repetitive firing as GKdr was increased. Modulating GKM primarily reduced firing rate across all GNav 1.8 values as GKM was increased.\n\nEach row of plots shows the results of changing one non-Nav 1.8 conductance to 0%, 50%, 100%, 150% and 200% of each model in the population’s original value and rerunning the simulation study design shown in Figure 4A. Only variation in GKdr and GKM show substantial effects on firing rate. Within each plot each square represents the mean number of AP/s fired during a step stimulus averaged across all models in the population.\n\nAs we found that GKM and GKdr levels each have an important role in determining the excitability of models in the population (Figure 6B), we ran a simulation study where we co-varied four parameters: stimulus amplitude; GNav 1.8; GKdr; and GKM, to see how changes in these three important conductances interacted.\n\nAs we were simulating changes in four parameters, we used a coarser range of values for each parameter compared to the previous simulation study design (Figure 4A). Stimulus amplitude was varied from 0 to 6000 pA in increments of 1000 pA and the conductance scaling factors for GNav 1.8, GKdr and GKM were each varied from 0.0 to 2.0 in increments of 0.5. All combinations of the four parameters over these ranges were simulated for a total of 875 simulations on each model in the population.\n\nFigure 8 shows the mean firing rate of the population in each of these simulations. As plots go from left to right, GKM increases, as plots go from bottom to top, GKdr increases.\n\nEach panel shows the results of simulations with a specific pair of GKM and GKdr scaling factors applied. Within each panel each square represents the mean number of AP/s fired during a step stimulus averaged across all models in the population with GKM, GKdr and GNav1.8 scaled by the relevant values. GKM increases in each panel from left to right, while GKdr increases from bottom to top.\n\nFour main trends can be seen in Figure 8:\n\n1. Increasing GKM generally decreases firing rate as can be seen by comparing the rightmost column (GKM = 2) to the central column (GKM = 1).\n\n2. Increasing GKdr increases firing rates at higher GNav 1.8 values but decreases firing rate at lower GNav 1.8 values – as can be seen by noting how in each column going from the bottom (GKdr = 0) to top (GKdr = 2) of that column the peak firing rate in each panel moves to the right (to higher GNav 1.8 values).\n\n3. Decreasing GKdr and GKM to very low levels almost complete prevents rapid firing – in the bottom left corner of the figure the mean firing rate in most simulations is close to 2 AP/s. This is primarily due to most models failing to repolarise. For example in the set of simulations where GKM and GKdr were set to 0 (bottom left panel, Figure 8), in every simulation where neither GNav 1.8 nor stimulus amplitude were set to 0 every model in the population exhibited repolarisation failure.\n\n4. When the sum of GKdr and GKM is low, decreasing GNav 1.8 from baseline can cause mean firing rate to decrease. This can be seen in many of the panels in the bottom left quadrant of Figure 8, for example in the case where GKdr = GKM = 0.5, decreasing Nav 1.8 by 50% causes a dramatic increase in firing rate at almost all tested stimulus amplitudes.\n\nTo better understand the relationship between the values of GKdr and GKM and their effects on firing rate, we looked at the overall trends for these conductances. We averaged mean firing rate over all of the GNav 1.8 scaling values we simulated. This condenses the information in each panel of Figure 8 down to a single averaged firing rate. We then plotted curves of either GKM or GKdr against mean firing rate averaged over GNav 1.8. This produced a group of curves (Figure 9) that show the general trend from varying GKdr (Figure 9A) or GKM (Figure 9B).\n\nLeft) Each line shows a different GKM scaling factor, with GKdr scaling factor varying on the x-axis.\n\nRight) Each line shows a different GKdr scaling factor, with GKM scaling factor varying on the x-axis.\n\nBroadly we see three different relationships between firing rate and the scaling factor of GKdr and GKM, depending on the scaling factor of the other conductance:\n\n1. Monotonically negative response - increasing the potassium conductance that wasn’t fixed always decreased firing rate – the expected response for a potassium current. This occurred when GKM or GKdr scaling factors were fixed at 1.5 and 2 (Figure 9A and B, red and purple curves).\n\n2. Monotonically positive response - increasing the potassium conductance that wasn’t fixed always increased firing rate. This is opposite to the expected response for a potassium conductance and occurs because without sufficient potassium current the models cannot effectively repolarize, which prevents repetitive firing. This only occurred when GKM was fixed at 0.\n\n3. Inflected response – increasing the potassium conductance that wasn’t fixed increased firing rate up to an inflection point, beyond which increasing the conductance further caused firing rate to decrease. This is the most common scenario.\n\nThese results shows that in our model, across a wide range of ionic conductance profiles, the response of firing rate to a change in a potassium conductance can qualitatively differ depending on the value of another potassium conductance. Therefore channel block by a drug could increase or decrease firing rate, depending on the ionic background of the hDRG neuron the drug was applied to.\n\n\nDiscussion\n\nIn this study, we investigated the roles the voltage-gated sodium channel Nav 1.8 and the IKdr and IKM potassium currents play in determining hDRG neuron excitability using a population of computational models calibrated against experimentally determined hDRG AP biomarker ranges. We used this population of models methodology to allow us to simulate the variability in ion channel densities found in real neurons and investigate how variability in many different ionic currents collectively interact to determine overall cellular excitability.\n\nThe main findings of this study were:\n\n1. We developed a new model of the hDRG neuron AP using published data from hDRG neurons at the AP level and at the ion channel level where data was available. We used this model as a baseline to develop a population of models that integrated experimentally observed variability in hDRG AP biomarkers.\n\n2. Altering the Nav 1.8 conductance had nonlinear effects on excitability. Decreasing the Nav 1.8 conductance from baseline in each model decreased the mean firing rate of the population, however increasing the Nav 1.8 conductance initially increased firing rate up to a point, beyond which firing rate decreased.\n\n3. Simulation studies with the population of models identified key roles for the M-type potassium current and delayed-rectifier potassium current. Increasing the M-type potassium current conductance decreased firing rate. However, increasing the delayed-rectifier potassium current increased firing rate, but only when Nav 1.8 was also increased by a similar factor.\n\n4. Several of our results agree with the literature on DRG neuron electrophysiology, which provides credibility for our modelling approach. Increasing Nav 1.8 conductance increased AP width, in line with current understanding of the role of human Nav 1.8. [Han et al., 2015]. Our finding that increasing M-type potassium channel conductance decreased excitability agrees with studies that show M-channel opening drugs such as retigabine reduce sensitivity to nociceptive stimuli in peripheral nerve fibres [Passmore et al. 2012, Vetter et al. 2013, Huang et al. 2016].\n\nThe baseline hDRG model we developed has several novel features that make it adapted to modelling hDRG neurons. We used ionic current models developed from recordings of human channels, for Nav 1.7, Nav 1.8 and Nav 1.9 [Vasylyev et al., 2014; Han et al., 2015; Huang et al., 2014]. It was particularly important to have a human-specific Nav 1.8 model as it has substantial differences in kinetics compared to rat Nav 1.8 [Han et al., 2015]. The kinetics of inactivation of human Nav 1.8 are slower, and the V1/2 of inactivation is more depolarized, so more human Nav 1.8 channels are available, for longer, during the action potential, compared to rat Nav 1.8. Previous models and most studies of DRG neuron electrophysiology have used rodent DRG neurons due to the difficulty of acquiring hDRG neurons for research. Therefore, species differences in AP morphology and ion channel behaviours [Han et al. 2015] were not captured.\n\nTo mimic inter-neuronal variability in ion channel conductances and AP biomarkers we used the population of models methodology [Britton et al., 2013] and experimental data from ex vivo hDRG AP recordings to calibrate the population of models to match the ranges of AP biomarkers from those experiments. Similar approaches to this have been used in many modelling studies of other neuron types (e.g. [Prinz et al., 2003, 2004]) and of cardiac cells (e.g. [Britton et al. 2013, Passini et al., 2017, Muszkiewicz et al., 2016]) but to our knowledge they have not previously been used in studies of DRG neurons.\n\nWhere hDRG neuron-specific data was not available we built on previous modelling studies of DRG neurons, in particular work by Tigerholm et al. (2015) and Choi and Waxman (2011). Tigerholm et al. investigated activity-dependent slowing in a model of a DRG neuron axon. As human-specific potassium current and hyperpolarisation-activated cation current models were not available we used the same potassium and h-current models that were used in this study. Choi and Waxman investigated how varying GNav 1.7 and GNav 1.8 affected excitability in a single compartment model of a DRG neuron cell body. We adapted the geometry and passive membrane properties used in this study for our model.\n\nComputational hDRG neuron models, along with other alternatives such as ex-vivo [Davidson et al., 2014] and stem cell-derived hDRG neurons, offer alternatives that could replace the use of rodent DRG neuron recordings in the future. For example, a computational model could be used to investigate how different ion channel modulating drugs interacted with a pathological ion channel mutation in hDRG neurons, to determine if they could reduce or counteract the pathological effects of the mutation. This type of study would require a large number of rodent DRG neurons if performed using an animal model, as the experiment would need to be repeated for each different drug tested. Computational models also have the advantage that they can be refined and iterated upon as new data becomes available, to make the most of the limited availability of hDRG neurons and recordings. Furthermore the fact that they are human-based allows for easier clinical translation, as animal moedels present patho-physiological differences with human cells, which can impact therapy outcomes.\n\nTo estimate the number of animals currently used in studies of rodent DRG neuron excitability, that could potentially be replaced by these alternatives, we searched PubMed to estimate the use of rodents in this class of experiment. Our base search query was: (mouse OR mice OR rat OR rodent) AND (nociceptor OR DRG OR dorsal root ganglion) AND pain AND (ion channel OR ion channels OR Nav) with the addition of:\n\n1) AND (drug block OR channel block OR pharmacological block OR current inhibition OR current block)\n\n2) AND (mutation OR channelopathy)\n\nThese queries aimed to find papers that investigated: 1) Drug-induced changes in human DRG excitability and 2) Changes in human DRG excitability caused by ion channel mutations, as these are the main areas that could be investigated with our population of models.\n\nThe base search produced 1018 papers published over the last 5 years (2015-20), indicating wide use of these models. Filtering further using Queries 1 and 2 reduced these papers to 361 unique non-review papers published between 2015 and 2020. We analysed the first 20 papers for each query to determine average numbers of animals used for DRG studies only. Mean animals used for DRG studies in the 16/40 papers where number of animals used was reported or could be inferred from figures was 103, with a range of 24 to 273. Therefore, annual animal use in these experiments is estimated at 7,400 animals (103 animals multiplier by 361 papers divided by 5 years), while total annual animal use in all DRG experiments is estimated at 21,000 animals (using the figure of 1018 papers found by the base query). These estimates do not include animal use in industry, which is likely to be substantial given the current interest in developing selective sodium channel blockers targeting human DRGs.\n\nChanges to ionic currents rarely affect only a single channel. For example, pathological changes in channel expression often affect multiple channels [Black et al., 2004; Cao et al., 2010; Zhao et al., 2013], and even selective ion channel blocking drugs often have non-negligible side effects.\n\nNav 1.8 is known to support repetitive firing in DRG neurons [Renganathan et al., 2001; Blair and Bean, 2002], due to its rapid recovery from inactivation [Dib-Hajj et al., 1997]. Therefore we expected that increasing GNav 1.8 would likely increase firing rate across the population. However, our simulations showed a more complex relationship. Increasing GNav 1.8 by up to approximately 30% increased mean population firing rate but beyond this inflection point increasing GNav 1.8 caused the mean population firing rate to decrease. We also found that the GNav 1.8 value at which this inflection point occurred was highly dependent on GKdr (Figure 9B). Enhancing or blocking GKdr caused the inflection point to move to higher or lower GNav 1.8 values respectively.\n\nThe opposing contributions of Nav 1.8 and IKdr during repolarisation of the action potential (Figure 3) suggest the balance between Nav 1.8 and IKdr may be important for supporting repetitive firing in hDRG neurons. However, as more ionic currents are expressed in real hDRG neurons than are included in our model, it is likely the true relationship will be more complicated than simply balancing GKdr against GNav 1.8.\n\nWe found that the conductance of the M-type potassium current, primarily carried by Kv 7.2, 7.3 and 7.5 in DRG neurons [Passmore et al., 2003], strongly modulated the excitability of the population of models. Increased GKM decreased mean firing rate of the population of models across a wide range of stimulus amplitudes and GNav 1.8 and GKdr scaling factors (Figure 8). This is in agreement with studies showing that M-current activators such as retigabine and flupirtine can reduce sensitivity of nociceptors to nociceptive stimuli [Passmore et al., 2012; Vetter et al., 2013; Huang et al., 2016]. The accepted role of the M-current is to stabilize the membrane potential against small depolarisations. Our results suggest that the M-current could also have a role in opposing large stimulus currents during sustained firing, due to the M-current’s slow kinetics of activation that lead to it reaching its maximum amplitude only after a period of repetitive firing (Figure 3).\n\nWe think that future development of the model presented here should focus on three areas: validation against new experimental data as they become available; incorporating additional ionic currents, particularly calcium-activated potassium currents and calcium currents; and expanding the geometry of the model to include the DRG neuron axon and t-junction with the cell body.\n\nThe population of models used in this study was calibrated using AP biomarker data from wild type hDRG neurons stimulated under standard conditions (e.g. no applied drugs). Adding additional calibration steps with AP biomarkers from experiments under very different conditions (e.g. under drug block) would further constrain the allowed conductance profiles of accepted models which should result in a population that behaves more realistically under a wider range of simulated conditions. This overcomes an important limitation from previous models, which are based in animal data.\n\nOur current baseline model contains eight ionic current models that cover a diverse range of sodium and potassium currents but do not include calcium currents, calcium-activated potassium currents, or ionic pumps and exchangers. All of these would be good targets to add to the model however there is less data, particularly DRG-specific data, to parameterise models of these currents, so this work would either require using models developed for other neuron types or new experimental data.\n\nExtending the population of models to include a realistic DRG neuron geometry would allow new research questions to be considered and could extend calibration of the population, for example models could be calibrated against the known range of conduction velocities in C-fibres. The variability in conductance could also be extended to account for differences in ion channel expression at different parts of the axon and cell body [Calvo et al., 2016; Tsantoulas and McMahon, 2014].\n\n\nData availability\n\nZenodo: Data supporting “Changes in human dorsal root ganglion neuron excitability from modulating Nav 1.8 conductance are non-linear and depend on the conductances of the delayed rectifier and M-type potassium currents: a simulation study”. https://doi.org/10.5281/zenodo.5512414\n\nThis project contains the following underlying data:\n\n• drg-pom-master.zip (Source code, models and examples for this study and drgpom software.)\n\n• Figure_1_data.csv (Voltage trace data underlying Figure 1A.)\n\n• Figure_2_data.csv (Conductance scaling factors for each model in the population of models, underlying Figure 2.)\n\n• Figure_3A_data.csv (Conductance scaling factors for the models in Figure 3.)\n\n• Figure_3B_model_[1/2/3/4/5/6]_data.csv (Voltage and current traces underlying Figure 3B.)\n\n• Figure_4A_data.csv (Simulation parameter data underlying Figure 4A.)\n\n• Figure_4BC_data.csv (Mean number of APs; AP half width data; and simulation parameters underlying Figure 4B and 4C.)\n\n• Figure_5_data.csv (Half width and AP firing rate data for individual models underlying Figure 5.)\n\n• Figure_6A_data.csv (Conductance and model category data underlying Figure 6A.)\n\n• Figure_6B_GKdr.csv (Mean number of APs and simulation parameters underlying the top row of Figure 6B.)\n\n• Figure_6B_GKM.csv (Mean number of APs and simulation parameters underlying the bottom row of Figure 6B.)\n\n• Figure_7_data.csv (Amplitudes, conductance scaling factors and firing rates underlying Figure 7.)\n\n• Figure_8_data.csv (Mean number of APs and simulation parameters underlying Figure 8.)\n\n• Figure_9_left_panel_data.csv (Mean firing rate and GKdr and GKM scaling factors underlying left panel of Figure 9.)\n\n• Figure_9_right_panel_data.csv (Mean firing rate and GKdr and GKM scaling factors underlying right panel of Figure 9.)\n\n• Equations_and_conductance_densities.docx (Equations and conductance densities for the baseline model.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license.", "appendix": "References\n\nAkopian AN, Sivilotti L, Wood JN: A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature. 1996 Jan; 379(6562): 257–262. Publisher Full Text\n\nAlonso LM, Marder E: Visualization of currents in neural models with similar behavior and different conductance densities. elife. 2019 Jan; 8(8): e42722. Publisher Full Text\n\nBlack JA, Liu S, Tanaka M, et al.: Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain. Pain. 2004 Apr 1; 108(3): 237–247. PubMed Abstract \| Publisher Full Text\n\nBlair NT, Bean BP: Roles of tetrodotoxin (TTX)-sensitive Na+ current, TTX-resistant Na+ current, and Ca2+ current in the action potentials of nociceptive sensory neurons. J. Neurosci. 2002 Dec 1; 22(23): 10277–10290. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBritton OJ, Bueno-Orovio A, Van Ammel K, et al.: Experimentally calibrated population of models predicts and explains intersubject variability in cardiac cellular electrophysiology. Proc. Natl. Acad. Sci. U. S. A. 2013; 110(23): E2098–E2105. PubMed Abstract \| Publisher Full Text\n\nCalvo M, Richards N, Schmid AB, et al.: Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury. elife. 2016; 5: e12661. PubMed Abstract \| Publisher Full Text\n\nCao XH, Byun HS, Chen SR, et al.: Reduction in voltage-gated K+ channel activity in primary sensory neurons in painful diabetic neuropathy: role of brain-derived neurotrophic factor. J. Neurochem. 2010; 114(5): 1460–1475. PubMed Abstract \| Publisher Full Text\n\nDavidson S, Copits BA, Zhang J, et al.: Human sensory neurons: Membrane properties and sensitization by inflammatory mediators. Pain. 2014 Sep 1; 155(9): 1861–1870. PubMed Abstract \| Publisher Full Text\n\nDib-Hajj SD, Ishikawa K, Cummins TR, et al.: Insertion of a SNS-specific tetrapeptide in S3-S4 linker of D4 accelerates recovery from inactivation of skeletal muscle voltage-gated Na channel mu1 in HEK293 cells. FEBS Lett. 1997; 416(1): 11–14. PubMed Abstract \| Publisher Full Text\n\nFaber CG, Lauria G, Merkies IS, et al.: Gain-of-function Nav1.8 mutations in painful neuropathy. Proc. Natl. Acad. Sci. U. S. A. 2012; 109(47): 19444–19449. PubMed Abstract \| Publisher Full Text\n\nFischer BD, Ho C, Kuzin I, et al.: Chronic exposure to tumor necrosis factor in vivo induces hyperalgesia, upregulates sodium channel gene expression and alters the cellular electrophysiology of dorsal root ganglion neurons. Neurosci. Lett. 2017; 653: 195–201. PubMed Abstract \| Publisher Full Text\n\nHan C, Estacion M, Huang J, et al.: Human Na(v)1.8: enhanced persistent and ramp currents contribute to distinct firing properties of human DRG neurons. J. Neurophysiol. 2015; 113(9): 3172–3185. PubMed Abstract \| Publisher Full Text\n\nHan C, Vasylyev D, Macala LJ, et al.: The G1662S NaV1.8 mutation in small fibre neuropathy: impaired inactivation underlying DRG neuron hyperexcitability. J. Neurol. Neurosurg. Psychiatry. 2014; 85(5): 499–505. PubMed Abstract \| Publisher Full Text\n\nHindmarsh AC, Brown PN, Grant KE, et al.: SUNDIALS: Suite of nonlinear and differential/algebraic equation solvers. ACM Transactions on Mathematical Software (TOMS). 2005 Sep 1; 31(3): 363–396. Publisher Full Text\n\nHodgkin AL, Huxley AF: A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 1952 Aug 28; 117(4): 500–544. Publisher Full Text Reference Source\n\nHuang D, Huang S, Gao H, et al.: Redox-dependent modulation of T-type Ca2+ channels in sensory neurons contributes to acute anti-nociceptive effect of substance P. Antioxid. Redox Signal. 2016 Aug 10; 25(5): 233–251. PubMed Abstract \| Publisher Full Text\n\nHuang J, Han C, Estacion M, et al.: Gain-of-function mutations in sodium channel Na(v)1.9 in painful neuropathy. Brain. 2014; 137(Pt 6): 1627–1642. PubMed Abstract \| Publisher Full Text\n\nHuang J, Yang Y, Zhao P, et al.: Small-fiber neuropathy Nav1.8 mutation shifts activation to hyperpolarized potentials and increases excitability of dorsal root ganglion neurons. J. Neurosci. 2013; 33(35): 14087–14097. PubMed Abstract \| Publisher Full Text\n\nJarvis MF, Honore P, Shieh CC, et al.: A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc. Natl. Acad. Sci. U. S. A. 2007; 104(20): 8520–8525. PubMed Abstract \| Publisher Full Text\n\nKouranova EV, Strassle BW, Ring RH, et al.: Hyperpolarization-activated cyclic nucleotide-gated channel mRNA and protein expression in large versus small diameter dorsal root ganglion neurons: correlation with hyperpolarization-activated current gating. Neuroscience. 2008; 153(4): 1008–1019. PubMed Abstract \| Publisher Full Text\n\nMaingret F, Coste B, Padilla F, et al.: Inflammatory mediators increase Nav1.9 current and excitability in nociceptors through a coincident detection mechanism. J. Gen. Physiol. 2008; 131(3): 211–225. PubMed Abstract \| Publisher Full Text\n\nMarder E, Taylor AL: Multiple models to capture the variability in biological neurons and networks. Nat. Neurosci. 2011; 14(2): 133–138. PubMed Abstract \| Publisher Full Text\n\nMcKay MD, Beckman RJ, Conover WJ: A comparison of three methods for selecting values of input variables in the analysis of output from a computer code. Technometrics. 1979; 21(2): 239–245.\n\nMeyer RA, Campbell JN: Myelinated nociceptive afferents account for the hyperalgesia that follows a burn to the hand. Science. 1981 Sep 25; 213(4515): 1527–1529. PubMed Abstract \| Publisher Full Text\n\nMuszkiewicz A, Britton OJ, Gemmell P, et al.: Variability in cardiac electrophysiology: Using experimentally-calibrated populations of models to move beyond the single virtual physiological human paradigm. Prog. Biophys. Mol. Biol. 2016; 120(1-3): 115–127. PubMed Abstract \| Publisher Full Text\n\nPassini E, Britton OJ, Lu HR, et al.: Human In Silico Drug Trials Demonstrate Higher Accuracy than Animal Models in Predicting Clinical Pro-Arrhythmic Cardiotoxicity. Front. Physiol. 2017; 8: 668. PubMed Abstract \| Publisher Full Text\n\nPassmore GM, Reilly JM, Thakur M, et al.: Functional significance of M-type potassium channels in nociceptive cutaneous sensory endings. Front. Mol. Neurosci. 2012; 5: 63.\n\nPassmore GM, Selyanko AA, Mistry M, et al.: KCNQ/M currents in sensory neurons: significance for pain therapy. J. Neurosci. 2003; 23(18): 7227–7236. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPayne CE, Brown AR, Theile JW, et al.: A novel selective and orally bioavailable Nav 1.8 channel blocker, PF-01247324, attenuates nociception and sensory neuron excitability. Br. J. Pharmacol. 2015; 172(10): 2654–2670. PubMed Abstract \| Publisher Full Text\n\nPrinz AA, Billimoria CP, Marder E: Alternative to hand-tuning conductance-based models: construction and analysis of databases of model neurons. J. Neurophysiol. 2003; 90(6): 3998–4015. PubMed Abstract \| Publisher Full Text\n\nPrinz AA, Bucher D, Marder E: Similar network activity from disparate circuit parameters. Nat. Neurosci. 2004; 7(12): 1345–1352. PubMed Abstract \| Publisher Full Text\n\nRatté S, Zhu Y, Lee KY, et al.: Criticality and degeneracy in injury-induced changes in primary afferent excitability and the implications for neuropathic pain. elife. 2014; 3: e02370. PubMed Abstract \| Publisher Full Text\n\nRenganathan M, Cummins TR, Waxman SG: Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons. J. Neurophysiol. 2001; 86(2): 629–640. PubMed Abstract \| Publisher Full Text\n\nRho YA, Prescott SA: Identification of molecular pathologies sufficient to cause neuropathic excitability in primary somatosensory afferents using dynamical systems theory. PLoS Comput. Biol. 2012 May; 8(5): e1002524. PubMed Abstract \| Publisher Full Text\n\nSangameswaran L, Delgado SG, Fish LM, et al.: Structure and function of a novel voltage-gated, tetrodotoxin-resistant sodium channel specific to sensory neurons. J. Biol. Chem. 1996; 271(11): 5953–5956. PubMed Abstract \| Publisher Full Text\n\nSchulz DJ, Goaillard JM, Marder E: Variable channel expression in identified single and electrically coupled neurons in different animals. Nat. Neurosci. 2006 Mar; 9(3): 356–362. PubMed Abstract \| Publisher Full Text\n\nSheets PL, Jackson JO, Waxman SG, et al.: A Nav1.7 channel mutation associated with hereditary erythromelalgia contributes to neuronal hyperexcitability and displays reduced lidocaine sensitivity. J. Physiol. Lond. 2007; 581(Pt 3): 1019–1031. PubMed Abstract \| Publisher Full Text\n\nSorensen SA, Bernard A, Menon V, et al.: Correlated gene expression and target specificity demonstrate excitatory projection neuron diversity. Cereb. Cortex. 2015; 25(2): 433–449. PubMed Abstract \| Publisher Full Text\n\nTigerholm J, Petersson ME, Obreja O, et al.: Modeling activity-dependent changes of axonal spike conduction in primary afferent C-nociceptors. J. Neurophysiol. 2014; 111(9): 1721–1735. PubMed Abstract \| Publisher Full Text\n\nTsantoulas C, McMahon SB: Opening paths to novel analgesics: the role of potassium channels in chronic pain. Trends Neurosci. 2014; 37(3): 146–158. PubMed Abstract \| Publisher Full Text\n\nVasylyev DV, Han C, Zhao P, et al.: Dynamic-clamp analysis of wild-type human Nav1.7 and erythromelalgia mutant channel L858H. J. Neurophysiol. 2014; 111(7): 1429–1443. Publisher Full Text\n\nVetter I, Hein A, Sattler S, et al.: Amplified cold transduction in native nociceptors by M-channel inhibition. J. Neurosci. 2013; 33(42): 16627–16641. PubMed Abstract \| Publisher Full Text\n\nZhao X, Tang Z, Zhang H, et al.: A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons. Nat. Neurosci. 2013; 16(8): 1024–1031. PubMed Abstract \| Publisher Full Text" }	[ { "id": "208690", "date": "13 Dec 2023", "name": "Dr. Gonzalo Hernandez Hernandez", "expertise": [ "Reviewer Expertise Ion channel and G protein-coupled receptor molecular modeling and simulations." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have reviewed the manuscript in detail and recommend it for approval with specific reservations. While the paper demonstrates considerable academic merit, there are minor modifications that, if addressed, could enhance the overall strength and impact of the article.\nSummary: This manuscript by Britton O.J and Rodriguez B. represents a comprehensive method article introducing an open-source software designed for simulating human dorsal root ganglion (hDRG) neuron electrophysiology. Initially, a baseline model with eight transmembrane currents representing sodium and potassium channels, was established, followed by the generation of a pool of candidate models using a population approach. This aimed to align them with specific experimental AP biomarker ranges. In addition, the research focused on the NaV1.8 channel conductance to elucidate its influence on neuronal excitability in relation to other ion channels in nociceptive neurons. Notably, modifications in the NaV1.8 conductance revealed an interesting behavior: a moderate increase heightened the firing rate, but an excessive increase reduced it. The study also underscored the importance of delayed rectifiers and M-type potassium conductances in determining neuronal excitability. In particular, perturbations in the delayed rectifier potassium conductance affected the NaV1.8's impact on the firing rate. The authors performed a detailed simulation analysis on the variation of those as well as other conductances on the action potential firing rates revealing interesting trends, which can be tested experimentally and, in the future, also used for the development of new specific ion channel modulators.\nStrengths: The manuscript is well written. The hypothesis is well-defined and the rationale for the study is clear, giving readers a good idea of what to expect in the subsequent sections. In addition, the methodology employed for capturing inter-neuronal variability is noteworthy.\nThe model accounts for variations in ionic conductances and action potential morphology, making it more comprehensive and likely more representative of biological reality.\nA major strength of the paper is the sharing of their software openly. This move makes the research more trustworthy and lets others build on it. Open tools can also help new researchers learn. Instructions for the use of software are very clear.\n\nAreas of potential improvement:\n\nBy focusing solely on the soma, the model may not capture the complete electrophysiological behavior of the entire neuron, especially any interactions between the soma and an axon. This is perhaps beyond the scope of the study and can be acknowledged as a study limitation.\nThe simplifying assumption that ion channel structures do not vary between different DRG neurons might not hold true for all neurons and conditions. There is ion channel structural variability, which may influence their open probabilities, gating time constants and other functional parameters. This assumption could impact the overall accuracy and applicability of the model and can be also mentioned as a limitation.\nI would also consider moving sections of the Methods, which discuss software installation, running etc. towards the end of the paper as an Appendix, in order not to break the flow of the paper.\n\nUpon a detailed evaluation of your manuscript, I appreciate the depth of the research presented. I believe that with certain refinements in the figures, Results, and Discussion sections, the manuscript will greatly benefit in its readability and overall impact.\nHere are my specific recommendations:\n\nFigure 1. The current name on the right got cut. Analysis: It remains ambiguous if these models were run until they achieved a particular steady-state and then fitted it to the mean values of each parameter based on the data recorded from ex vivo hDRG neurons. For Figure 2, utilizing smaller data points and a more distinguishable color palette could enhance the clarity of the distribution of conductances across the population. Figure 3, The \"currentscape\" visualisation technique implemented in Figure 3 offers a unique insight into the conductances and relative current magnitudes across six models. To further its effectiveness: Opt for color schemes that offer greater contrast to discern the various currents more distinctly. Incorporate a dynamic repetition of action potentials to enrich presentation. Figure 4. I am not sure if panel A is needed, as the same information is already present in panels B and C, as the same range and individual data points as squares are shown there. Although, I do see that Fig. 4A is referenced multiple times for other data as well. Figure 5. top panel. No action potential (AP) firing is visible. Maybe, an inset with zoomed in data on this panel would be helpful. Figure 6. What is the difference between dark- and light-red plots in panel A? Figures 7 & 8: Both figures summarise the principal trends recognized in the study. To augment the easiness of their interpretation: Enhance the legibility of the x and y axes, the axes are hard to read as the figure contains multiple panels. Figure 9 Colors for GKM/GKdr 0 and 2 as well as for 0.5 and 1.5 are hard to distinguish. While the procedure involving the stimulation of different levels of block and enhancement of NaV1.8 conductance is mentioned, a clearer description of the methodology could be helpful. This includes specifics about how 'different levels' were defined and chosen. Currently it is not clear whether they (as well as other conductances) were selected solely based on physiological interneuron variability, action of a pharmaceutical or another modulator or both. The NaV1.8'’s influence on hDRG neuron excitability analysis could benefit from a section discussing potential future investigations or implications of these findings on possible therapeutic interventions, given the evident role of NaV1.8 in neuronal firing. The comparison and usage of previous modeling studies of DRG neurons, particularly from Tigerholm et al. and Choi and Waxman, adds depth to the study. It would be advantageous to highlight any adaptations or modifications made to these referenced studies' methodologies and the reasoning behind these changes. Throughout the paper: It would be best not to use space in the \"GNav 1.8” and similar variables. Ideally one would also use subscript for channel name, i.e., \"G_{Nav1.8}\". Ideally, voltage-gated ion channel names should include subscript capital “V” and not “v” as well. Please briefly explain the word \"rheobase\", which is specific to the ion channel electrophysiology field and may not be known to a general reader. Please explain what IKdr current is. There is no link for \"Choi and Waxman, 2011\" paper. \"Each cells containing code\" -> \"Each cell containing code\" \"kleak ion channel model \" -> \"leak K^+ channel (Kleak) model\"? \"simulating changes in multiple conductances at multiple different levels\" - Do you mean levels associated with certain changes of conductances from the base model or something else? \"For GNav 1.8, this is because models with low GNav 1.8 have peak voltages that are below the experimental range, and models with high GNav 1.8 have threshold voltages that are below the experimental range.\" - please check if this is correct as stated, as it seems that both low and high \"GNav 1.8 \" have the same effect. \"combination of three biomarkers: threshold voltage, peak voltage and AP full width parameters\". Are these parameters? I thought they were properties or simulation outputs. \"\"That GKdr is actually larger in the rapidly firing group suggests it is not just a lack of total potassium current conductance, and a corresponding lack of outward current to oppose repetitive firing, that causes these models to fire so rapidly.\" This sentence is confusing, please clarify if possible. Maybe, the comma after \"conductance\" causes this confusion. \"Mean animals used for DRG studies in the 16/40 papers where number of animals used was reported or could be inferred from figures was 103\". Please reword this sentence as it is confusing from the start. What does \"mean animals\" mean?\nConclusions: The authors have done an excellent job in pointing out areas for potential improvements in their model. Adding more details about calcium-related currents and pumps and transporters will make the model even better. The authors also did not mention chloride channels, which are known to be present in DRG neurons as well (see e.g., Wilke et al Front Neurosci. 2020; 14: 287). Also, showing the full structure of hDRG neurons, such as the connections between axons and the main cell body (soma), will make the model more realistic. I look forward to seeing how the authors will refine their work in future updates and how they will extend their population models to capture hDRG neuron variability in human datasets available in the future.\n\nAre a suitable application and appropriate end-users identified? Yes\n\nAre the 3Rs implications of the work described accurately? Yes\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "208694", "date": "14 Dec 2023", "name": "Stewart Heitmann", "expertise": [ "Reviewer Expertise Computational cardiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study uses computational modelling to investigate how the human dorsal root ganglion (DRG) neuron responds when the Nav 1.8 sodium channel is selectively modulated. The study is motivated by the role of the DRG neuron in nociception (pain) and the role of the Nav 1.8 channel in modulating the excitability of that neuron. A population of models is used to investigate phenotypically diverse DRG neurons drawn from the human population. They find that the excitability of the neuron depends not only on Nav 1.8 but also on potassium ion channels which appear to be naturally co-regulated with Nav 1.8.\nThe proposed model offers practical benefits in reducing the use of animals in pain research, which I applaud. The population of models approach is useful for addressing the issue of natural variation too.\nMethods\nA substantial part of the manuscript is dedicated to instructions for installing and running the software. Documenting the software is important, but those issues are separate from the scientific question that is being investigated (about ion channels). So I urge the authors to move all of the software-related text (pages 7-13) into a separate software manual or tutorial paper. Software documentation is best shipped with the source code.\nDiscussion\nUpon reading the discussion, I felt no wiser about how co-regulated INav 1.8 and potassium currents might inform better treatments for patients with pathological pain. The discussion of the M-type potassium current seemed to hint that it might it be possible to treat a defective Nav 1.8 ion channel by targeting the potassium channels. I would have like to see that sort of thing explored in greater depth.\nMinor Comments\npg 3. Suggested rewording:\n“For example, a gain-of-function mutation in Nav 1.7 caused hyper-excitability in DRG neurons that expressed Nav 1.8, yet the same mutation caused hypo-excitability in DRG neurons that lacked Nav 1.8 {Rush et al., 2006]”\npg 4. “… with eight action potential parameters.” Biomarkers is a better word than parameters.\npg 4. “In this study, we use a non-standard modelling approach … “. Is it really non-standard? Perhaps say instead: “In this study, we use a modelling approach based on experimentally calibrated …”\n\npg 4. “We developed a baseline model …”\nI suggest moving Figure 1B here to support the text.\np5. “The equations for the baseline model can be found in the supplementary material”.\nPersonally, I like to see the equations included in the main text, if only in abbreviated form, so that the reader can appreciate what is being modeled. However, that is not always practical.\np6. “Each model was simulated …”\nIt would be nice to plot the stimulus protocols to spare the reader from looking it up. It is also worth describing how the randomized models were equilibrated prior to being analyzed. Some AP models can take hundreds of beats to equilibrate to a new parameter regime.\np14. “This correlation is caused by calibrating on …”\nI’m not sure this is the best wording. Instead of saying the correlation is caused by the calibration (which is to say that it is caused by your method), I suggest saying that the correlation reveals the normal relationship between ion channels (as caused by nature).\nFigure 3A. The a-h labels on the abscissa are slightly confusing. Better to label them as GNav17, GNav18, etc.\nFigure 4A is unneccesary. You could omit it altogether. The idea is clear enough from panels B and C. That said, the effect of Nav 1.8 scaling could be made even clearer by plotting it for only one stimulus amplitude, say 4000pA.\np18. “We hypothesised that one reason for the decrease in firing rate …”.\nIllustrating the idea with some example AP trains would be helpful.\np25. “To estimate the number of animals currently …”\nThere is no need to explain the details of the pubmed search, just summarize it. For example: “To estimate the number of animals that could be potentially be replaced with our proposed DRG model, we searched pubmed for all papers in the past five years that described pain research using DRG neurons in rodents. We estimated that 21,000 animals were used annually for this purpose. These estimates do not include animal use in industry”.\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? Partly", "responses": [] }, { "id": "230124", "date": "06 Mar 2024", "name": "Markos Xenakis", "expertise": [ "Reviewer Expertise Computational Neurophysiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present work, the authors, Oliver J. Britton, and Blanca Rodriguez, make a decisive step toward whole-hDRG-neuron modeling and simulation. They are motivated by the need for more realistic insilico models for studying the transmission of nociceptive stimuli. They systematically investigate how inter-neuronal ion channel density heterogeneity contributes to the shape of hDRG action potential. This is done in a computationally efficient manner without sacrificing biophysics realism. Namely, the authors introduce a pool of hDRG models where model-to-model variability of ion channel conductances but not ion channel kinetics is implemented. Their modeling procedures are documented pedagogically. They emphasize the role of the NaV1.8 channel in shaping action potential properties. Specifically, they report on a balancing mechanism regulating neuron firing rate concerning an inflection point of the NaV1.8 conductance. Driving NaV1.8 conductance above its inflection point value decreases the neuron firing rate. The location of the inflection point is reported to depend on the delayed rectifier potassium conductance. The authors conclude that synergies among the NaV1.8 and other channels, especially potassium, play an essential role in modulating the NaV1.8 channel in nociceptive neurons.\nThe study paves the way for the replacement, refinement, and reduction of animals in research. The presented model shows good potential in reproducing hDRG electrophysiological signatures. It is constructed comprehensively and transparently based on previous works and experimental data. What I missed, however, is a knowledge gain in neuropathic pain disease. Remarks and comments for improvement are attached below.\nMajor comments:\nMethodological aspects:\nThe authors do a great job documenting their procedures and sharing step-by-step guidelines on installing and running the software. However, all this occupies too much space in the manuscript, which could be used for other, more relevant purposes, such as explaining more in-depth how presented findings can help better understand neurophysiological aspects of nociception. I hence suggest that text and tables from p. 7 “Installing drgpom and compiling models .. ” to p. 13 “.. . This example can be used as a template to design your own simulation studies.” to be placed elsewhere. For example, the authors could consider the option of moving this material to the corresponding Zenodo and/or GitHub repository.\n\nResults:\nOn a few occasions, it is unclear whether findings reflect specific parameter choices or actual neurophysiological traits. A better explanation of the relationship between the former (parameter space) and the latter (neurophysiology) could help. Specifically:\n“For Gnav 1.8, this is because models with low GNav 1.8 have peak voltages that are below the experimental range, and models with high GNav 1.8 have threshold voltages that are below the experimental range. For GKA, no model with high GKA had a combination of threshold voltage, peak voltage, and after-hyperpolarisation time constant that was simultaneously within the experimental ranges of these three AP biomarkers.” - What is the neurophysiological rationale in support of these simulation results?\n“This correlation is caused by calibrating on a combination of three biomarkers: threshold voltage, peak voltage and AP full width parameters, as removing all of these AP biomarkers from the calibration process also removes the correlation between GNav 1.8 and GKdr and removing any one of them reduces the correlation coefficient. This indicates that in our model the balance of these two currents is important throughout the action potential, from upstroke (threshold, peak voltage) to repolarisation (AP full width).” - What is the principle underlying reported correlation? Is it a computational artifact, or does it reflect a biophysically interesting phenomenon?\n\n“The relative proportions of these currents are however highly variable from model to model. Similarly, the main currents during the upstroke are INav 1.7 (red) and INav 1.8 (blue), but some models have a large contribution by Nav 1.7 and in some models the contribution is very small, while the contribution of INav 1.8 is uniformly large.” - Could the authors clarify how NaV1.7 and NaV1.8 contribute differently to the current generation?\n\nConclusions and Discussion:\nBased on the abstract and keywords, the reader expects to learn something about neuropathic pain disease. After reading the manuscript, however, this expectation still needs to be met. The role of the NaV1.7, which has emerged as a key regulator of neuropathic pain disease, could also be addressed, especially concerning the pivotal role NaV1.8 is suggested to have here. Could the authors elaborate on how presented findings and modeling procedures enhance our understanding of neuropathic pain?\n\nMinor comments:\nOn the first page, in the Methods description section, a full stop is missing: “.. in ionic conductances and action potential morphology” should read “.. in ionic conductances and action potential morphology.”\np. 6: Please provide SI units of the time constant \\tau\n\np. 9: “Temperature in Celcius” should read “Temperature in Celcius”\n\np. 12: “Davidson et al., 2014 PAIN are supported”. Is “PAIN” a typo here?\n\np. 13: “.. consisted of 328 models .. “. Where does the number 328 comes from?\n\np. 14: “For GNav 1.8, this is because models with low GNav 1.8 have peak voltages that are below the experimental range, and models with high GNav 1.8 have threshold voltages that are below the experimental range.\" This is unclear and needs clarification (see also major comment above).\n\np. 16: “As we show later, the balance of these currents is not as critical as the balance of GNav 1.8 and GKdr at rheobase, where only a single AP is fired.” Please clarify what “critical” means in this context.\n\nFigure 4A: Is this illustration really necessary?\n\np. 22: “.. almost complete .. “ should read “.. almost completely ..”\n\np. 24: “Similar approaches to this have been used .. ” should read “Similar approaches have been used ..”\n\np.24: Tigerholm et al. (2015) and Choi and Waxman (2011) hyperlinks do not work.\n\nAre a suitable application and appropriate end-users identified? Yes\n\nAre the 3Rs implications of the work described accurately? Yes\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "176219", "date": "16 Sep 2024", "name": "Joseph J Pancrazio", "expertise": [ "Reviewer Expertise microelectrode arrays", "electrophysiology", "nociceptors" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 created a novel approach to the utilization of Hodgkin-Huxley based models for excitable cells to interpret how pharmacological manipulation of underlying channels affects excitability. They do so by creating a diverse \"family\" of permutations from a central model and parse the population by accepting those models that yield overall action potential behavior that offers parameters within the observation range of prior experiments. The authors focus on modeling the pharmacological inhibition of a particular channel type, Nav 1.8, which is implicated in the manifestation of chronic pain, and therefore, serves as a potential target for drug discovery.\nThe approach is innovative yielding insight into the entanglement of voltage dependent ion channel activity and the action potential behavior that emerges and is also a function of that very ion channel behavior. The paper is well-written, the results are informative, yielding insight into degrees of channel inhibition that may -- just may be -- the right target level for effectiveness. I have relatively few comments for this innovative and impressive work:\n1. Fig. 4A showing a uniform grid with dots appears to be entirely uninformative. It appears to be simply indicating that there are 328 qualified models. I suggest removing Fig. 4A. 2. \"Implications for the 3Rs ''' section in the discussion. While well intentioned, the literature analysis which attempts to arrive at some number of animals lost in unnecessary experimentation is a weak argument. It seems to suggest that animal experimentation could have been eliminated if this modeling approach was adopted without reservation or further validation. While I am an optimist for the utility of modeling, the notion that the job is entirely done is premature. I suggest this section be eliminated and simply make the point that modeling of this nature has the potential to substantially reduce the animal subject burden during preclinical testing (as do hIPSCs for that matter).\n\nAre a suitable application and appropriate end-users identified? Yes\n\nAre the 3Rs implications of the work described accurately? Yes\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-104
https://f1000research.com/articles/11-103/v1	27 Jan 22	{ "type": "Case Report", "title": "Case Report: Large bone defect reconstruction in lower extremities with ipsilateral transposition fibular graft with combined inlay and onlay technique using plate and screw fixation (fibula pro tibia technique)", "authors": [ "Ananto Satya Pradana", "Krisna Yuarno Phatama", "Edi Mustamsir", "Irasiqin Wibawanto S", "Lasa Dhakka Siahaan", "Mohamad Hidayat", "Respati Suryanto Dradjat", "Krisna Yuarno Phatama", "Edi Mustamsir", "Irasiqin Wibawanto S", "Lasa Dhakka Siahaan", "Mohamad Hidayat", "Respati Suryanto Dradjat" ], "abstract": "Introduction: Management of large bone defect is a challenging problem. Hahns, in 1884, introduced the first use of fibula pro tibia to bridge a large defect of the tibia due to chronic osteomyelitis. In this case, we did a transposition of the ipsilateral fibular with inlay and onlay technique using a locking plate and screw into the defect of the tibia. Case presentation: A 20-year-old male came to our emergency department at RSUD Dr. Saiful Anwar, Malang, Indonesia with an open fracture grade III B of his left lower leg. We found a large defect of the tibia approximately 7.5 cm after regular wound care for ten months. Then, we performed ipsilateral transposition fibular graft with combined inlay and onlay technique using a locking plate and screw fixation. No infections occurred and there was progressive callus formation with extending ossification along the periosteal tissue in the four-month postoperative evaluation. There was no leg length discrepancy, and the union sign showed that the patient could achieve full range of movement (ROM) and walk with crutches without pain with a Lower Extremity Functional Scale (LEFS) score of 62. Discussion: Contralateral fibula graft carries a high risk of endangering the unaffected limb. The ipsilateral fibula can be utilized to replace the missing segment with minimal morbidity. The purpose of combining ipsilateral fibular transport with the inlay and onlay technique is to minimalize the gap defect between fracture fragments, therefore enhancing the union rate of the bone. Conclusion: The fibula pro tibia technique can be used as an alternative treatment option for large gap bone defects in lower extremities with minimal complication.", "keywords": [ "large gap bone defect", "transposition fibular graft reconstruction", "inlay and onlay technique", "fibula pro tibia", "case report" ], "content": "Introduction\n\nLarge segmental bone defects of the tibia are challenging for both surgeon and patient.1,2 Segmental bone defects are most commonly caused by fractures from high energy trauma, osteomyelitis, benign or malignant bone tumor surgery, septic nonunion, and congenital abnormality.2–4\n\nA free vascularized fibula graft is commonly used because it is mechanically strong, retains the intrinsic blood supply, osteogenic, and can be used for large bone defects in various places.4,5 However, there are certain drawbacks of free vascularized fibula grafts, such as donor side morbidity (unaffected limb), peroneal nerve injury, and the surgery necessitates a microsurgery technique, thus requiring a long operating time.2,3 In 1884, Hanh described an alternative technique using the ipsilateral vascularized fibula as a graft for a 12 cm segmental tibia defect due to chronic osteomyelitis in an eight-year-old male. Instead of cutting the fibula and performing a reanastomosis at a distance, Hanh simply transposed the fibula with his pedicle into the tibial defect.3,4,6\n\nIpsilateral vascularized fibula transfer (IVFT) allows the surgeon to transfer the fibula to the tibia as a complete graft without disrupting soft tissue attachment, blood supply and without needing microsurgical techniques, resulting in bone healing at both ends of the defect.1,3,4\n\nIn this case, we transposed the ipsilateral fibular with inlay and onlay technique using a locking plate and screw into the tibia gap defect in a one-stage procedure followed with serial radiographic X-ray evaluations and functional outcome evaluation using the Lower Extremity Functional Scale (LEFS) scoring system.7 This report has followed the CARE and Surgical CAse REport (SCARE) checklist and guidelines.8 In addition, written informed consent was obtained from our patient for publication of their data and clinical images.\n\n\nCase report\n\nA 20-year-old male came to our emergency department at RSUD Dr. Saiful Anwar Malang Indonesia with an open fracture grade III B of his left lower leg (Figure 1). The cause of the injury was a high-speed motorcycle collision. The patient did not consume any routine medication and did not have any other illness. We performed debridement, serial irrigation and applied an external fixation device on the affected leg (Figure 2). After regular wound care for ten months, the soft tissue condition improved, but we found a nonunion of the tibia with a large defect of approximately 7.5 cm (Figures 3 and 4). We planned to perform external fixation removal with a reconstruction of the bone defect using an ipsilateral transposition fibular graft with a combined inlay and onlay technique using a locking plate and screw fixation (fibula pro tibia technique).\n\nBefore the operation, we did a thorough preoperative evaluation that included measuring the extent of the bone defect and assessing the quality of the adjacent soft tissue and joints. A thorough excision of all avascular bone back to bleeding tissue was performed through an anterolateral approach. The peroneal nerve and its branches were identified and protected. The fibula is dissected from its surrounding soft tissue, keeping muscles and periosteum to protect the periosteal vascularization. The dissected fibula was moved to the posterior interosseus membrane of the tibial defect with the preserved vascularization without tension on the soft tissues. Then, the graft was fixed proximally and distally with a locking plate to improve stability. Meticulous care was taken during the transpose to avoid kinking or stretching of the vascularization. In addition, we performed an onlay technique using an ipsilateral avascular fibular graft fixated with screws to provide more mechanical strength and minimize the gap defect between fracture fragments, therefore enhancing the union rate of the bone (Figures 5 and 6). To ensure ankle stability, at least 10 cm of the distal fibula must be preserved. After receiving intravenous antibiotics for one week, the patient was allowed to go home. His left leg was braced and weight-bearing was prohibited until the vascularized bone graft healed and, periodically, the patient was followed up clinically and radiographically (Figure 7). No infections occurred in the four-month postoperative evaluation, and progressive callus formation with extending ossification along the periosteal tissue was seen in serial radiographic X-ray evaluations. Clinically, the LEFS score was 62 (good), there was no leg length discrepancy, and the union sign showed as the patient could achieve full range of movement (ROM) and walk with crutches without pain.\n\n(A, B) One month. (C, D) Three months. (E, F) Four months.\n\n\nDiscussion\n\nLarge segmental tibial defects have been treated successfully using a variety of procedures, including autogenous corticocancellous bone grafting, tibiofibular synostosis, ipsilateral vascularized fibula transfer (IVFT), allograft tibial reconstruction, free vascularized fibula transfer, and bone transport accompanied with Ilizarov technique.1–3 Proximal transtibial amputation is one of the options of treatment, but amputation should be avoided if the foot and ankle vascularization is normal.1 In addition, for most patients, shortening of limbs or amputation is an unacceptable condition.3,9\n\nBone transfer or corticocancellous bone grafting can be used to treat shorter defects of up to 5 cm. In contrast, larger tibial defects usually need a more complicated repair procedure, such as bone transfer or a free vascularized fibula transfer.3,9 The optimal treatment would provide appropriate vascularization as well as the availability of the essential osteoinductive, osteoconductive, and osteoprogenitor components. It should also allow for early mobilization while reducing the possibility of leg length discrepancy or axial deformity.3\n\nA variety of vascularized grafts are available for treatment. Vascularized bone grafts from the fibula or iliac crest have been applied for large lesions with satisfactory functional results. However, the iliac crest can only be used to treat defects 10–15 cm long, and the anatomical aspects of the iliac bone pose a congruency issue when replacing tubular bone such as the tibia and pose a high rate of donor morbidity, primarily pain and incisional hernias.2,3\n\nThe fibula is a suitable graft material because of its long, straight cortical bone that can bridge most defects, good structural strength, osteogenic potential, does not cause distant donor site morbidity and, unlike allografts, it has no immunogenicity, thus making the fibula a popular donor site for long bone defects.1,5,10 The fibula also bears just 6–15% of the weight transmitted via the leg, and it is considered expendable.4 The size of the fibular graft and its straight configuration allows it to fit into the femur or tibia medullary canal, allowing restoration of significant defects up to 26 cm in length.3,5\n\nFibula grafts can be harvested from the ipsilateral or contralateral limb. However, contralateral vascularized or non-vascularized fibula transfer carries a high risk of endangering the unaffected limb. The risk of donor-site morbidity and microvascular thrombosis should always be taken into account. Deep infection, peroneal nerve damage, long operation time, and contralateral unaffected limb ankle instability are all detrimental complications.2 Infection, rejection, fracture, and nonunion have also been reported with these procedures.1,9\n\nThe fibula possesses dual vascularity, with endosteal and periosteal vessels, and this is preserved in fibula pro tibia and provide a firm mechanical and biological framework for union.1,5 Experiments on Macaca monkeys showed that a vascularized pedicle graft of the ipsilateral fibula's shaft could be placed across a tibia defect and remained alive even when separated from the surrounding tissue.1,4\n\nThe main benefit of a perfused transplant is that the biological potential of living bone is preserved. In vascularized grafts, the osteocytes and other osteoprogenitor cells are maintained.11 Therefore, the grafts take less time to consolidate, have more remodeling potential, are more resistant to infection, and have better long-term mechanical characteristics. In addition, unlike an allograft, it has no immunogenicity.1,4,5,10 The vascularized bone graft maintains its mass and architecture better than an avascular fibular graft, is biomechanically stronger and has better healing potential and hypertrophy. Furthermore, in scarred and avascular recipient locations, the vascularized bone transplant provides a significant source of vascularity.5\n\nMore benefits of a perfused transplant are that primary or secondary bone healing is used to integrate viable grafts rather than creeping substitution because the vascularized bone graft skips the creeping substitution process. Creeping substitution process characterized by graft necrosis, resorption, and new bone growth in avascular transplants.5,10,11\n\nIn a study by Föhn et al., the ipsilateral fibula was used as a bone graft and positioned into the proximal and distal medullary canal of the fractured site with its peroneal and periosteal vascularization.9 The technique used in this case was the same, but we performed an additional onlay technique using an ipsilateral avascular fibular graft to provide more mechanical strength to the injury site. Moreover, the purpose of combining ipsilateral fibular transport with inlay and onlay technique is to minimize the gap defect between fracture fragments, therefore enhancing the union rate of the bone. Meanwhile, the downside of this technique is the need for a large incision to do the operation.\n\nWe use a locking plate to do the internal fixation because a locking plate is a type of internal fixator that combines the benefits of external fixation techniques with biological plating technique into one unit. Therefore, the lesion is stabilized, reducing interfragmentary motion and inflammation and providing a better environment for graft incorporation and bone union.5\n\nMechanical stress or stress loading on bone is widely acknowledged as an essential aspect in maintaining a proper balance between bone formation and resorption. Adaptive response in which bone formation outpaces resorption can occur when mechanical stress on long bones is increased. However, if the external mechanical loading is greater than the bone's strength, a stress fracture will occur.10 We planned gradual weight bearing with serial radiography follow-up to avoid stress fractures until the bone graft had hypertrophied sufficiently before total weight-bearing.\n\nFöhn et al. accomplished a single-step fibula pro tibia procedure with no contralateral limb morbidity because they used ipsilateral fibula and less operation time because the graft vascularization was intact, thus micro anastomosis was not required.9\n\nIn this patient, an infection on the surgery site was not seen. This outcome was also found in the study of Koulouvaris et al.3 He reported that the average infection rate was 2.1 percent in ipsilateral vascularized fibula transfer papers, albeit this varied depending on the number of osteomyelitis patients treated. In an average of 8.5 percent of cases, the fibula graft fractured, but a sound union was achieved within six months, and patient mobilization and the outcomes were described as good in the majority of cases.3\n\nThe fibula originally takes only one-sixth of the leg's static load. However, the fibula will grow if it is subjected to higher loading forces.1 In the fibula pro tibia, the fibula undergoes hypertrophy and becomes an integral part of the static supporting architecture of the leg when it is subjected to more than usual weight-bearing loads.2 Föhn et al., also described that the periosteum of the remaining fibula stumps also played a significant role in neo-ossification in fibula remodeling into a tibia-shaped dimension.9 Gayito et al., in their study, reported that the bone remodeling process was observed with the gradual growth of the transferred fibula in the fibula pro tibia postoperatively. In their study, compared to the unaffected fibula, the diameter of the transferred fibula increased substantially by at least twice its initial size in eight years of observation.2\n\nThe weaknesses of the fibula pro tibia technique are that this technique cannot be done for tibia defects that are very proximal or distal. The fibular graft can only be moved a certain distance without disrupting its vascularization and the disrupted vascularization can make the graft avascular.1,4 Moreover, when implanted on an avascular and scarred bed, these avascular transplants are doomed to fail because if the union of the allograft is not achieved, a vascularization is impossible, and healing will never occur.4 It is also important to ensure the preservation of 8–10 cm or the distal fibular length to maintain ankle stability and cause no substantial ankle morbidity.12\n\nThe ‘fibula pro tibia’ technique is an inexpensive, simple, and efficient method compared to allografts. The advantages of ‘fibula pro tibia’ include the transfer of a living autograft with remodeling capability, infection resistance, and better long-term mechanical qualities.2\n\n\nConclusion\n\nIpsilateral transposition fibular graft reconstruction with a combined inlay and onlay technique using plate and screw fixation (fibula pro tibia technique) can be used as an alternative treatment option for large gap bone defects in lower extremities with minimal complication.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nShafi R, Fragomen AT, Rozbruch SR: Ipsilateral fibular transport using Ilizarov-Taylor spatial frame for a limb salvage reconstruction: a case report. HSS J. 2009; 5(1): 31–39. PubMed Abstract \| Publisher Full Text\n\nGayito RC, Priuli G, Traore SY, et al.: Treatment of large diaphyseal bone defect of the tibia by the “fibula pro tibia” technique: application in developing countries. Acta Orthop Belg. 2015; 81(1): 17–22. PubMed Abstract\n\nKoulouvaris P, Theos C, Kottakis S, et al.: A simple treatment for a 15-cm tibia bone defect: a case report of an ipsilateral vascularized fibula transfer. J Orthop Trauma. 2007; 21(3): 215–218. PubMed Abstract \| Publisher Full Text\n\nShapiro MS, Endrizzi DP, Cannon RM, et al.: Treatment of tibial defects and nonunions using ipsilateral vascularized fibular transposition. Clin Orthop Relat Res. 1993; 296: 207–212. Publisher Full Text\n\nSun Y, Zhang C, Jin D, et al.: Free vascularised fibular grafting in the treatment of large skeletal defects due to osteomyelitis. Int Orthop. 2010; 34(3): 425–430. PubMed Abstract \| Publisher Full Text\n\nRahmansyah N, Ariandi M, Yurianto H, et al.: Ipsilateral transposition fibular graft for reconstruction of the tibial diaphysis and soft tissue defect. Natl J Med Res. 2019; 9(1): 57–58.\n\nBinkley JM, Stratford PW, Lott SA, et al.: The Lower Extremity Functional Scale (LEFS): scale development, measurement properties, and clinical application. North American Orthopaedic Rehabilitation Research Network. Phys Ther. 1999; 79(4): 371–383. PubMed Abstract\n\nAgha RA, Franchi T, Sohrabi C, et al.: The SCARE 2020 Guideline: Updating Consensus Surgical CAse REport (SCARE) Guidelines. Int J Surg. 2020; 84: 226–230. PubMed Abstract \| Publisher Full Text\n\nFöhn M, Bannasch H, Stark GB: Single step fibula-pro-tibia transfer and soft tissue coverage with free myocutaneous latissimus dorsi flap after extensive osteomyelitis and soft tissue necrosis--a 3 year follow up. J Plast Reconstr Aesthet Surg. 2009; 62(11): e466–e470. PubMed Abstract \| Publisher Full Text\n\nLiu SJ, Lo WJ, Ueng SWN: Stress analysis of the vascularized fibular bone transplantation in large tibia defect reconstruction: A finite element study.2012; 13(5): 218–225. Publisher Full Text\n\nRoberts TT, Rosenbaum AJ: Bone grafts, bone substitutes and orthobiologics: The bridge between basic science and clinical advancements in fracture healing. Organogenesis. 2012; 8(4): 114–124. PubMed Abstract \| Publisher Full Text\n\nHong RG, Li R, Hu Y, et al.: Treatment options for infected bone defects in the lower extremities: free vascularized fibular graft or Ilizarov bone transport?. J Orthop Surg Res. 2020; 15(1): 439. PubMed Abstract \| Publisher Full Text" }	[ { "id": "121381", "date": "28 Feb 2022", "name": "R. Andri Primadhi", "expertise": [ "Reviewer Expertise orthopaedics and traumatology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article that would be suitable for indexing. The article provides a good treatment alternative for a wider hospital setting, not only in a fully equipped hospital, but also in a smaller hospital. It offered another perspective on open fracture with large bone gap cases. One of the accepted techniques for this kind of case is bone transport which is technically demanding and risk the patient's comfortability. This article proved that a simple external fixation and subsequent conversion is still efficacious.\nThe author should better describe the patient's peri-operative characteristics, including neurovascular status, smoking habit, diabetes, and so on, since these aspects may interfere with the results. Also, regarding the external fixator device, the author should mention why the custom made (using acrylic fixator) was applied, rather than more simple connecting rods and clamps.\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": "172854", "date": "12 Jun 2023", "name": "Luong Van Nguyen", "expertise": [ "Reviewer Expertise Orthopaedics and Traumatology", "limb lengthening." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 express sincere gratitude to get an opportunity to review the manuscript and congratulate the authors for such nice work. Reconstruction of large tibial defects could be done by various methods including segmental allograft, vascularized and non-vascularized autograft, induced membrane technique, and bone transport. The submission proves that external fixation of open tibial fracture and subsequent conversion of internal fixator using an ipsilateral transposition fibular graft with a combined inlay and onlay technique is efficacious. Microvascular skills and special devices were not required.\nHowever, there are some scopes for its improvement.\nIn the discussion, the author should discuss using external fixation. Why did the author not use ordinary external fixators?\n\nWhy was the tibial reconstruction performed 9 months after the injury?\n\nThe authors should discuss more disadvantages of the fibula pro tibia technique: It can not be applied in case of too large tibial defects, leg length discrepancy, or bad condition of leg soft tissue. The complications of this technique should be discussed.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-103
https://f1000research.com/articles/11-96/v1	26 Jan 22	{ "type": "Research Article", "title": "Sonification of weather data as a non-human-centric artistic approach", "authors": [ "Yi Kee Ng", "Kok Yoong Lim", "Yi Kee Ng" ], "abstract": "Background - In the mid-20th century, the emergence of sound studies demonstrated a shift of research interest in sonic practitioners. This field gains its prevalence by expanding the boundaries of prevailing conception through proposing alternative creative approaches in sound art practices.\nMethods – Two methods were presented – listening and sounding to promote creative sound making. The first method, listening involves soundwalking and recording sound from the external environments. These recordings were then re-evaluated and post-processed in audio editing software. The second method, sounding involves the creation of a weather data sonification system in Pure Data environment, in which the perceptual experience from the first method was taken into consideration.\nResult – First method enables the genesis of creative idiosyncrasies, such as preferences and ideas through the sonic perception of environmental events. In this process, noise and weather were identified as environmental components that share similar sensible qualities. Thus, noise is a prevalent medium that inspires the creation of sound generators in the sonification system presented in this paper. The sonic output of data sonification reveals an analogical connection between weather data and sonic parameters, in which changes in data values result in changes in acoustic properties. These outputs deliver different sensibilities based on their data parameters; sonification of temperature data might suggest an alarming effect to the listener.\nConclusion – The proposed methods were intricately linked, suggesting environmental events to be perceived and realized through a non-scientific perspective. By highlighting aesthetic possibilities of environmental components, this paper presents an alternative perspective in contrast with the human-centric worldview through the creation of sonic works.", "keywords": [ "Non-human-centric perspectives", "sonification", "sound design", "sonic parameters", "weather data" ], "content": "Introduction\n\nIn the 21st century, sound studies research1 prevailed as an academic field to investigate and reflect on the continuous establishment of strategies, culture, and aesthetics, amid the emergence of new sonic practices. The research was categorized into different tropes which focused on sonic perception, sonic sites and soundscapes, sonic reproduction, artists and collectives, and sonic aesthetics.\n\nTherefore, the strategies and creative possibilities of sonic methods under the category of soundscape were investigated. In other literature, the term soundscape was defined as ‘events heard, not objects seen’, whereas in this research it was defined as a discipline that examines the effects of the acoustic environment on the creatures and entities living within it. This then contributes to the establishment of various noise abatement and musical practices as methods of advocating and appreciating the beauty of environmental soundscape.2 Although soundscape research started as a musical endeavour for appreciation of environmental sound, which proclaimed the world as a ‘macrocosmic musical composition’, it only focused on collecting and examining ‘non-polluted’ sound through field recordings. Thus, this contradiction offers the possibilities of sonic practitioners to extend and practicalize its original concepts into other creative practices.\n\nDespite this, soundscape research encouraged sonic practitioners and non-practitioners to engage with the environment through listening. Instead of focusing on sound from the natural environment, works from avant-garde practitioners explore the creative possibilities of sound indiscriminately from different environments. In Sonic Meditations,3 the composer Oliveros encouraged a series of sonic experimental activities such as listening and recording environmental sounds, journaling listening experiences and producing sound works as part of deep listening practice. On the contrary, Feld’s acoustemological approach derived from a non-human perspective- to acknowledge relation shared with numerous human and non-human actants, and ultimately the construct of the world through ‘sonic ways of knowing’.4,5\n\nOn a similar notion, this paper will engage and discuss human and non-human relations through sonic means. We propose two methods – listening and sounding. First, listening and soundwalking were conducted in order to observe and examine the overlap of sonic components emitted from human and non-human influences in the physical environment. In hopes to better understand other non-sonic events of the environment, weather data was collected through a virtual open-source platform. Second, sounding involves analysing, post-processing and re-evaluating sound samples that were recorded per se. The findings of the analysis enable relation to be drawn between sonic properties and quality, which then guide the process of data sonification.\n\n\nMethods\n\nListening in the context of this paper draws closely on the methods that were promoted in previous deep listening practices.6 This involved observing and recording sonic events simultaneously when navigating through the sonic landscapes of different environments. In short, this method of perceiving defined soundwalking practice.\n\nThe process of soundwalking first involved observing and listening attentively to a different sound and sonic events that took place in the surrounding environment. This process was facilitated by using a digital audio recorder (Zoom H1n) which enables sound with different properties to be heard and detected as audio signal input. In other words, this facilitation intensified the listening experience, in which sound with lower amplitudes and high frequencies that were less audible, such as forest ambience, bird songs were amplified and heard through the device’s integrated stereo microphones. Thus, based on the properties of sound, the gain of signal inputs was monitored and adjusted accordingly.\n\nThen, soundwalking proceeded with identifying and recording environmental sound with desirable sensible qualities, specifically surrounding ambience that was often perceived as ‘noise’ or ‘insignificant sound’. Based on the understanding that sound possessed distinctive qualities in different spatiotemporal settings, soundwalking was conducted in both natural and urban environments. Decisions such as moving towards or away from the sound source were made on-site based on the changing qualities of the environment. Each recording was recorded in 5-7 minutes, thus durationally these recordings can be perceived as sonic events that documents the multiplicities of sonic components emitted from non-human and human producers.\n\nSoundwalking enables environmental events to be perceived primarily through sonic means, however, these events can also be perceived through other technological means. Hence, we identify weather as events that share similar sensible qualities with ambience recordings, that of being ‘unpredictable’ and ‘indeterminate’. In order to examine the changing process of weather events on a specific timeline, weather data of the soundwalking site was collected through a virtual open-source platform known as Open Weather Map (OWM) that integrates several data sources such as numerical weather prediction (NWP), weather stations and satellite data.7 The data was retrieved in numerical format through Application Programming Interface (API) calls and stored externally in comma-separated values (CSV) files, as shown in Figure 1. Hence, weather data collection can be seen as an extension of soundwalking – a method of environmental engagement.\n\nRecordings of the sonic events were categorized based on the details of recording sites, sonic components and sources. For example, sound recordings of forests were categorized as the natural soundscape that consists of bird song, wind noise and hums, whereas sound recordings of cityscape were categorized as the urban soundscape that consists of noises of transportation and traffic as some of the major sonic components. These details were documented to ensure future navigation.\n\nIn order to narrow down the variables of recordings, this paper only focuses on the sound that shares similar sensible qualities with the environment. The sound emitted from other sources such as birds, people and transportation were not sampled or used for analysis. Hence, the sound of the surrounding ambience such as the noise of wind and other geophysical influences were sampled out from the recordings into a shorter duration. These sampled sounds were most often perceived as ‘background noise’ or rumbles, in which the sources of emission were arbitrary or unrecognizable. To examine the sensible qualities of background ambience, the acoustic properties of different samples were compared through audio analysis tools, namely a frequency and amplitude follower.\n\nWe selected samples from both urban and natural environments; the comparison is better visualized in the form of spectrograms. The values of amplitude (dB) and frequency (Hz) were annotated on each spectrogram. The analysis was done with Sonic Visualizer. The figures below depict the result of acoustic analysis across a specific duration: Figure 2 shows a higher fluctuation of both amplitude and frequency as compared to Figure 3. The comparison of acoustic properties was summarized in Table 1.\n\nThe difference in acoustic properties result in the difference of sensible qualities: samples of cityscape could be perceived as loud, chaotic and noisy, whereas samples of forest could be perceived as soothing, calming and unobtrusive. These identified qualities enable the genesis of idiosyncratic preferences, knowledge and sensitivity on the sensibilities of noise. Thus, it indirectly inspires and influences the decision-making process of creating sound generators in the sonification process.\n\nSound generators were created in Pure Data (PD) – an open -source visual programming environment to sonify different parameters of weather data that were collected previously. The sonification process translates numerical data into sonic outputs - in which sonic parameters of generators were modified by incoming data parameters.8 Each data point maps to the frequencies and amplitudes as shown in Table 2. Consequently, this mapping allows the changes of data points to be revealed and perceived across time.\n\nTo distinguish weather data parameters from one another, the parameters were assigned to three different sound generators which were synthesized to deliver different sensible qualities. In order to represent the distinctive changes of temperature data, a sawtooth wave oscillator with an amplitude envelope of short release time was used to deliver a sharper sonic quality. In contrast, to represent the fluid and irregular physical quality of clouds, a sine wave oscillator and white noise generator were used; enhanced with slight reverberation to ensure a smoother sounding quality. Lastly, rain volume was represented with white noise generators by structures of high-pass and low pass filters to mimic rainy ambience. Figure 4 shows examples of sound generators in the sonification system.\n\nTo afford control in the process of sonification, the system was designed with a few interactive parameters. The front-end of the system was organized into two parts as shown in Figure 5, the top part display control parameters for input weather data, which consist of two options of data sets and controllable data reading intervals (milliseconds), whereas the bottom part display volume control (dB) for sonic output. In consequence, the sonic output varies each time when the control parameters were modified.\n\nFor a desirable result, the control parameters were set constant. Daily data was chosen from the dataset and its reading interval was set to 350 milliseconds, which means each data point was read and translated into sound between the given time intervals. Weather data parameters, namely temperature, cloud coverage and rain volume were sonified and exported separately into three (.wav) files with a duration of 3 minutes. Each sonification output was later visualized in spectrogram to draw a relation between weather data and sonic parameters.\n\n\nResults\n\nIn this paper, we propose sonic methods as practical ways of perceiving and re-enacting sensibilities of environmental events. This proposition presupposes events derived from everyday reality are possible catalysts for artistic creation. Through practicalizing these methods, a relation was drawn between environmental components and intricacies that were perceived, both through sonic and non-sonic means. In the first method, noise, ambience, and weather were identified as specific components that share similar sensible qualities, that of being unpredictable and random in its materialities. That being the case, the noise was highlighted as a prevalent sonic medium that inspires the creation of sound generators which were later used to represent the sensibilities of weather data through sonification.\n\nEnvironmental perception enables the formation of idiosyncratic experience, ideas, preferences, and affords ways of realizing and re-enacting sensibilities. In the second method, sonification was seen as a method in realizing and re-enacting sonic experiences. This method considers how weather events were experienced, for example, how temperature changes seemingly deliver an alarming effect, or how moving clouds might suggest a sense of wonder. Thus, each data parameter was mapped onto different sound generators to deliver distinctive sensibilities. Sonic output of temperature data was heard as distinctive changes in frequencies; cloud data was heard as subtle changes in frequencies, random changes in amplitude of noise; and rain was heard as ambience based on its occurrences. The outputs suggest an analogical connection between weather data parameters and sonic parameters, this connection is visualized in spectrograms, as shown in Figures 6, 7 and 8.\n\nThe spectrograms suggested the most distinctive changes in the acoustic properties of temperature sonification as compared to others. This method of outputting sonification process enables weather data to be represented as distinctive parameters. However, the output altered if all processes were outputted at once, the combination of different sonic qualities will generate a new sonic event; abstracting the changes in data values, thus blurring the relation between the parameters.\n\nUltimately, both methods were found to be intricately linked to one another in the processes of creating sonic works. The transition from one method to another is found to be fluid and interchangeable as these methods consist of other undiscussed variables. These uncertainties can be further explored as creative avenues.\n\n\nDiscussion/conclusions\n\nThis paper suggested that environmental events can be perceived from a non-scientific perspective in creative practice.\n\nThese methods enable knowledge, experience and sensibilities of the environment to be delivered through the creation of sonic works.\n\nIn the process of creation, the noise and weather events were highlighted as results of non-human geophysical influences. As such, noise was used as an aesthetic component to represent the irregularities of weather events. By tapping into the emancipatory aesthetic possibilities of noise, this paper offers an alternative way of environmental learning in contrast with the romantic environmentalist vision as prioritized in previous soundscape research.\n\nIn other respects, sonification was highlighted as a technical process that blurs the boundaries between scientific and artistic representation, in which relationships could be drawn in between. Sonification was a method proposed to realize the sensibilities of weather data; it also possessed the possibilities to generate new sensibilities and understanding each time when the process was executed. In contemporary sound art practice, sonification could be a useful method to reflect and apprehend the uncertainties of environmental event crises.\n\n\nFuture works\n\nThis paper only discusses limited scopes of non-human perspectives. With the flexibility of the proposed methods, we foresee the discussion and exploration to be stretched out to other types of quantifiable environmental data.\n\n\nData availability\n\nGithub: Underlying data for ‘Sonification of weather data as non-human-centric artistic approach’, https://github.com/yikee95/weather-data-sonification\n\nThis project contains the following data:\n\n• Pd-patch\n\n• CSV-file.pd\n\n• CSV-parse.pd\n\n• Main.pd\n\n• Save-audio-2.pd\n\nDistribution License is GPL3\n\n\nAuthor contributions\n\nNg Yi Kee: Conceptualization, Investigation, Methodology, Data Curation, Software, Visualization, Writing – Original Draft Preparation;\n\nLim KokYoong: Funding Acquisition, Supervision, Validation, Writing – Review and editing", "appendix": "Acknowledgements\n\nWe would like to thank Multimedia University for providing this opportunity and support for us to publish this study.\n\n\nReferences\n\nNardi C: The Sound Studies Reader (ed. Jonathan Sterne). Dancecult. 2013; 5: 80–85. Publisher Full Text\n\nSchafer RM: The Soundscape: Our Sonic Environment and the tuning of the world. Destiny Books; 1993.\n\nOliveros P: Sonic Meditations. Smith Publications; 1974.\n\nNovak D, Sakakeeny M: Keywords in sound. Duke University Press; 2015.\n\nLatour B: Reassembling the Social: An Introduction to actor-network-theory. Oxford: OUP; 2007.\n\nOliveros P: Deep listening: A composer’s sound practice. iUniverse; 2005.\n\nOpenWeatherMap.org. (n.d.). Accuracy and quality of weather data. OpenWeatherMap. Retrieved January 13, 2022. Reference Source\n\nHermann T, Hunt A, Neuhoff JG: The sonification handbook. Logos Verlag Berlin; 2011." }	[ { "id": "124361", "date": "24 Mar 2022", "name": "Tin Oberman", "expertise": [ "Reviewer Expertise soundscape research", "environmental acoustics", "landscape architecture", "spatial audio." ], "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 text features a description of an art project about sonification of weather data, written to fit the usual structure of a research article. With some modifications it could be a technical specification, but more modifications would be needed to become a research article. The technical specification could be a valuable addition to the body of knowledge on sonification.\nWhile I found the artistic approach intriguing, while writing this review, I didn't try to assess its artistic value but simply look at its scientific soundness. I'm afraid I wasn't able to recognise a valid research question, nor a scientifically sound connection between the results presented and the conclusions, hence the above mentioned recommendation about the technical specification format.\nThe literature used is scarce. The introduction is very general and doesn't provide enough context for the work presented in the methods and results sections. The details provided are not sufficient to ensure reproducibility and there are mistakes in the analysis and data interpretation (dB values in Figures 2 & 3 and Table 1). Steps laid out in the methods section are arbitrary, as is characteristic for an art work. Adding a reverberation effect to the part of the algorithm dedicated to sonification of clouds is a good example of this.\nThe purpose of mentioning the 'non-human-centric artistic approach' in the title is unclear.\nThe Abstract would need to be significantly improved.\nBackground doesn't seem to address the specific topic of the paper. The term 'sounding' seems a bit vague and it is unclear whether there are two or just one method as they feed into each other and the work presented doesn't appear to be valid without both. The abstract should be saying much more about what non-human-centric perspectives are and why they would be important. The role of sonification in this needs to be clearly described. Moreover, it seems that a lot of focus has been given to human perception throughout the work (choosing the timbre of the oscillators and deciding on how they will be modulated), one could even say that the sonification of weather data was clearly driven by human perception, so the non-human-centric part becomes even less clear.\nThe second paragraph of the introduction doesn't seem to take into account the body of soundscape research that very broadly evolved in the last 15-20 years around the interdisciplinary work relating to the ISO 12913 series. I have suggested some papers that could then lead into several soundscape-related directions1234.\nThe background described is very general and doesn't provide enough specifics related to the main work reported in this paper. I would suggest reshaping the introduction so it very clearly points out to the aims behind the main work reported. The key would be to describe other similar projects and reasons behind choosing this specific approach and methodology. Only the work by two composers is reported but activities such as listening and recording sounds and soundwalking go much beyond musical composition and this should be acknowledged. Further, as this seems to be the key, more space should be allowed to describe the non-human actants and how are they engaged in the soundscape framework.\nThe description of the Zoom H1n is vague and doesn't provide sufficient technical detail. Was the authors' intention to say that the recording doesn't accurately capture the dynamic range as experienced by the operators on-site? They should say so in much clearer words.\n\nMoreover, the methods should be described much more clearly. Currently it seems that the recording took place before soundwalking which doesn't seem to be possible. Perhaps a diagram would be helpful.\nWho perceived the surrounding ambience as noise and insignificant? The researchers? Was that a sampling strategy?\nThe details of where and when the soundwalks took place would be crucial to ensure replicability.\nPerhaps there is no need to describe checking the environmental data online as an 'environmental engagement'. Monitoring and collecting weather data is a usual part of any soundwalk as per ISO/TS 12913-2.\nHow was the categorisation performed? Please elaborate in much higher detail.\nA spectrogram combining amplitude and frequency would be much more useful in Figures 2 and 3.\nThe y scales in Figures 1 and 2, as well as the Table 1 look wrong, the dB values are impossibly small, the data should be checked.\nPure Data is indeed a open-source visual programming environment that can be used for sonification but more precision would be needed here to describe the specific patch that was used for the task in question. It doesn't seem like it is properly referenced.\nThe coding applied in the Table 2 seems arbitrary, and while a certain musical and perceptual logic was given in the following paragraphs, it is not clear how those decisions serve the purpose of the paper. Further, white noise is a very harshly sounding waveform per se so it is not clear why it was used with a sine wave oscillator and why for instance low pass filtering was not considered in the design. Instead of adding reverberation, perhaps different attack and decay/release values could have been considered to enhance the perception of smoothness. Perhaps a complex delay processor could have been considered. This all only points out how arbitrary the process was. I would suggest to change the narrative to the point where all the artistic actions wouldn't be calling to be put under scrutiny but taken as they were, while the focus of the paper could become its potential implementation, perception or similar, if the authors do not decide to take the path of a technical specification/report.\nThe vague points in the results and conclusions - how exactly was noise highlighted as a prevalent sonic medium inspiring the creation of sound generators? What kind of a relation was drawn between environmental components and (which?) perceived intricacies? Was there any indication that environmental events couldn't be used in a sound-related practice? While weather might not be entirely predictable, it surely can be predicted in certain time series. For instance, if we can forecast daily or weekly weather condition with some certainty, why were the 5-7 minute recordings considered to be relevant to depict the irregularity of the weather? Keeping in mind the body of soundscape research available in the recent years, I am not certain why are the authors referring to it as a 'romantic environmentalist vision'.\n\nFigure 4 is low quality and not all important parts are visible.\nEnglish language is excellent.\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? No", "responses": [] }, { "id": "202619", "date": "12 Sep 2023", "name": "Maxime Poret", "expertise": [ "Reviewer Expertise computer science", "sonification" ], "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\nNote: I am more of a \"scientific sonification\" person and, admittedly, a lot of the artistic considerations explored in this article tend to go over my head, especially as they pertain more to soundscape design than sonification. I would recommend getting an \"artistic sonification\" reviewer involved instead.\nThe abstract is hard to follow. The authors give some very broad background context, then announce two methods without first explaining what purpose they are meant to fulfill.\nThe first part of the results announcement makes enough sense (some relations exist between environmental sounds and weather), but I am confused as to what has been proved in the second half, with regards to the sonification of weather data (as the phrasing just reads like a basic definition of sonification).\nSome more context may need to be given as to what \"non-human centric\" means in the context of this paper, and how it relates to the methods being employed.\nI don't quite understand what makes field recordings \"non-polluted\", or how it contradicts the original ambitions of soundscape research.\nRather than \"methods\", the two experiments reported in this paper seem like they should be called \"phases\" of the same experiment, since one is clearly used as a basis for the next.\nThe description of the listening/soundwalking/data collecting phase could use a bit more detail: how many people were soundwalking? Over how many sessions? For what lengths of time? How much data does that amount to in total?\nThe sonification mapping proposed here seems valid enough, but there needs to be some explicit justification as to how the sonic parameters were chosen and how those choices relate to the observations made on the data collected in the previous phase.\nDespite announcing an interest in using noisy sounds to provide an understanding of the environment, it appears that a lot of the sounds used in the sonification are tonal in nature?\nThe results section really sounds like it is saying \"our (seemingly arbitrary) mapping of variables to sounds shows that there is a perceptual/cognitive relation between those variables and those sounds\", which I really hope is not the intended takeaway, as it makes as much sense as saying \"I colored this strawberry blue, which proves that strawberries are blue\".\nWas there some form of testing done, involving actual listeners, in order to determine that the data-sound mappings made sense? Or is that part of the non-human approach?\nThe supplementary materials should include some audio demos of the sonification.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "202616", "date": "22 Sep 2023", "name": "Massimo Grassi", "expertise": [ "Reviewer Expertise Experimental 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\nThis article investigates new strategies and creative possibilities in the understanding and utilization of soundscapes. While the manuscript undeniably presents a novel approach, I would reject the manuscript as in its current form it is not, in my opinion, suitable for indexing. There are several substantial areas that, in my perspective, require significant refinement. I would recommend reconsideration following a rigorous revision to address the current concerns.\n\nThere's ambiguity about the paper's intended audience, with some sections seemingly addressing scientific sonification experts while other sections lean towards artistic sonification. Catering the manuscript towards a more specific audience would facilitate a clearer conveyance of the central arguments and contributions of the work.\nThe abstract could offer a more detailed preview of the methodological approaches undertaken in the study, as currently it provides broad context without specifying the purpose of the methods announced. Amplifying the clarity here would set a structured pathway for the readers to follow, ensuring a smoother navigation through the paper.\nThe introduction could be expanded with great benefit for the readers. Clarifying terminologies such as “non-human centric” and “non-polluted” field recordings could foster a deeper understanding of the context of the study. Additionally, enhancing the range and depth of the literature referenced could potentially offer a more robust groundwork for the research. In particular, the use of the specific “soundscape” seems to be disconnected from the available literature, as the authors themselves point out. Knowing more about the reasons behind authors’ choices would be interesting.\nIn my opinion, the paper could also benefit from better articulating the reasons behind the chosen methodology. The two \"methods\" seem to be interconnected, however the connection is not clear. The research questions, even if in the context of an explorative research, are not adequately explicated. Further details on the listening/soundwalking/data collecting phase, such as participant numbers and session durations, are necessary for understanding and replicability. The sonification mapping lacks explicit justification for sonic parameter choices and their relation to the observations made. The sequence of methods, like the timing of recording versus soundwalking, is unclear. As a result, reproducibility is compromised due to insufficient details about the soundwalks. The process employed in the paper, particularly regarding some sonification choices like the use of white noise and reverberation, seems arbitrary. A more throughout explanation about the reasons of the authors choice would resolve this issue.\nMoreover, I perceive a certain dichotomy in the approach; while advocating a non-human-centric stance, the study seems to significantly lean on human perception in various aspects. An elucidation of this apparent contradiction could potentially enrich the discussion and contribute to a more cohesive narrative.\n\nIn the section discussing the results, striving for a more precise depiction of the relationships explored and the observations highlighted, notably on how noise was highlighted and the relationship between environmental components and perceived intricacies, could add to the efficacy of the narrative.\nOverall, while the attempt to bridge the artistic and scientific realms is commendable, the paper might find a more potent expression through substantial revisions targeted at enhancing clarity and depth. An alternative path might be reframing the narrative towards a technical specification or focus on its potential implementation and perception, or focusing more deeply on the artistic ramifications of sonification by better exploring the connections with human/non-human relations through sound, and delineating the knowledge and experiences facilitated through this sonic creation, along with identifying the potential beneficiaries.\nI know my review will disappoint the authors, but I hope that this feedback will assist in refining the manuscript to spotlight the significant contributions of this research in the fields of soundscapes and sonification.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "175751", "date": "13 Sep 2024", "name": "Balandino Di Donato", "expertise": [ "Reviewer Expertise Sound Design", "Interactive Audio", "Music", "Sonic Interaction Design." ], "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\n- There are some inconsistencies.\nThe abstract talks about the mid-20th century but then the introduction is contextualised in the 21st century.\n- Missing references.\n\"In other literature, the term soundscape was defined as ‘events heard, not objects seen’,\" - what literature do you refer to? Also I believe there is a grammar mistake. I should be written, In others’\n\n\"Although soundscape research started as a musical endeavour for appreciation of environmental sound, which proclaimed the world as a ‘macrocosmic musical composition’\" - who proclaimed it? With what work?\n\nThe introduction section is confusing and each paragraph seems a standalone text. I would encourage the authors to link better each concept.\nTo be clarified:\nIt is unclear that is the research aim of this paper. It buried in between the lines. There should be a cleared definition of the aims of this work in the introduction section.\n\n\"Soundwalking enables environmental events to be perceived primarily through sonic means, however, these events can also be perceived through other technological means.\" - What other technological means? To what do you refer to?\n\n\"In order to narrow down the variables of recordings, this paper only focuses on the sound that shares similar sensible qualities with the environment\" - What are these qualities? What is the definition of sensible qualities?\n\nThe type of audio synthesis utilise is unclear. What method was uses? FM? AM? Additive synthesis? The PD patches do not tell much about the audio synthesis. A diagram with the synthesis details is much more useful to the reader. Is the audio synthesis a pure choice of the author? Is there any study on the type of synthesis employed? What was the process/reason that led to this choice?\n\nIn the last paragraph of the method section the author talk about daily data. So, what is the relationship between the soundscape recording collected on a specific day with specific weather conditions, and the weather data?\n\nThe discussion and results section there is no presence audio example of these sounds. As the research is about soundscape, providing sound examples is vital for the full appreciation of the work.\n\nThere are different aspects that still needs covering and clarification. Soundwalks and sonification are two approaches that have been used for decades. There is nothing novel in this. There is something new in the approach to synthesise perhaps, and the context in which these approaches are used. However, this paper needs further clarification.\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? No", "responses": [] } ]	1	https://f1000research.com/articles/11-96
https://f1000research.com/articles/11-95/v1	26 Jan 22	{ "type": "Research Article", "title": "‘The great publication race’ vs ‘abandon paper counting’: Benchmarking ECR publication and co-authorship rates over past 50 years to inform research evaluation", "authors": [ "Dave Kendal", "Kate E. Lee", "Kylie Soanes", "Caragh G. Threlfall", "Kate E. Lee", "Kylie Soanes", "Caragh G. Threlfall" ], "abstract": "Background: Publication and co-authorship rates have been increasing over decades. In response, calls are being made to restrict the number of publications included in research evaluations. Yet there is little evidence to guide publication expectations and inform research evaluation for early career researchers (ECRs).\nMethods: Here we examine the early career publication and co-authorship records between 1970 and 2019 of >140,000 authors of 2.8 million publications, to identify how publication and co-authorship rates have changed over the last 50 years. This examination is conducted in order to develop benchmarks of median publication rates for sensibly evaluating ECR research productivity, and to explore success in meeting these benchmarks with different co-authorship strategies using regression models.\nResults: Publication rates of multidisciplinary ECRs publishing in Nature, Science and PNAS have increased by 46% over the last 50 years and that publications rates in a set of disciplinary journals have increased by 105%. Co-authorship rates have increased even more, particularly for the multidisciplinary sample which now has 572% more co-authors per publication. Benchmarks based on median publication rates for all authors increased from one publication per year at the start of a career, to four publications per year after 10 years of publishing, and one first-author publication across all years. The probability of meeting these benchmarks increases when authors publish with different co-authors, and first authorship rates decrease for ECRs with many co-authors per publication.\nConclusion: This evidence could be used to inform sensible publishing expectations for ECRs and the institutions they work for, and to inform calls to limit the number of publications produced by researchers and those used in research evaluations.", "keywords": [ "publication metrics", "publish or perish", "academic careers", "early-career researchers", "postdocs", "ECRs" ], "content": "Introduction\n\nPublication metrics are commonly used for managing academic expectations and evaluating research performance, both by researchers and the institutions they work for.1 Despite widespread concerns about their use and abuse,1–3 publication counts, citations and impact factors are still commonly used to guide academic hiring and promotion decisions, and to allocate funding resources.4–6 Yet there is increasing concern that a focus on such metrics has negative consequences, both for science and the researchers themselves. An unbalanced focus on volume may come at the expense of other important aspects of scientific endeavour, such as quality, engagement, and impact. A reward system that incentivises publication quantity can reduce research quality7,8 as the focus shifts to researcher status rather than knowledge gain,1 leading to “unsustainable science”.9 In response, calls are being made to “abandon paper counting”,3 limit the number of publications used in research performance evaluations3 and even limit the number of papers published by researchers.10 Yet this debate about increasing publication rates, and an appropriate response by researchers and those who evaluate them, has largely ignored early career researchers (ECRs).\n\nThere is potential that this unsustainable increase in publication rates could be amplified amongst ECRs, particularly as commonly used publication metrics do not fairly describe their performance; forcing ECRs to focus on publication counting. For example, the widely-used h-index is poorly suited to evaluating early-career research11–13 and it can take many years for high quality research to be reflected in an increase in an author’s h-index. Predictors of academic ‘success’ are often difficult for ECRs to control; publishing earlier,14–16 working at a more prestigious institution,5 or publishing with top scientists17 are outside of the control of most ECRs. In the context of uncapped publication expectations, the primary goal for many ECRs is to publish as many papers as possible: “Four papers are better than three. And five are better than four”.18 However, favouring quantity of publications is a potentially maladaptive behaviour. It is a poor predictor of later research ‘quality’ as measured by indicators such as citation rates.19 Research evaluation that defaults to publication counting will also disadvantage ECRs with families, those who teach, those who engage with industry or the broader public, or those who work in smaller labs; as they balance producing publications with a variety of competing responsibilities.20,21\n\nThere is currently little empirical data to make sense of these debates for ECRs, or for the research institutions and grant assessors evaluating their research performance. We know that ECRs are publishing earlier compared with ECRs from previous decades, often before their PhD is complete.22 Yet it is not clear that the pressure to publish is actually leading to increased publication rates for current ECRs, compared with previous generations. Publication strategies that could be potentially useful for ECRs and those who evaluate their research performance, have yet to be tested for ECRs. For example, strategies such as publishing with a large number of co-authors14,23 or in a range of different titles.23,24 Calls to severely restrict publishing or the number of publications used in research evaluation3,10 may diminish the pressure to publish, but could be counterproductive if applied to ECRs who are busily developing networks and skills through publications.\n\nA research evaluation framework that is specific to the early years of a career is needed. This should be based on an understanding of how ECR publication and co-authorship rates have changed through time, how they progress during a career, and what strategies best support research ‘success’. It should remove the pressures generated by uncapped publication expectations, and at the same time it should not unnecessarily restrict publication activity, which could inhibit collaboration and skill development. It should recognise the changing patterns of co-authorship, from large team-based publications to sole authorship. This study aims to generate empirical data to support the development of a research evaluation framework suitable for ECRs by:\n\n1) Characterising how publication rates for ECRs have changed over the last 50 years.\n\n2) Developing evidence-based benchmarks for ECR publication rates as careers develop.\n\n3) Exploring the effect of co-authorship strategies on the probability of researchers meeting these benchmarks.\n\n\nMethods\n\nTwo author samples were selected. The first (multidisciplinary) sample was selected to gain insights into publishing and co-authorship trends between 1970-2019 in the broader field of science, particularly high-ranking international journals that are so often used as an indicator of career success. This included all authors who published in the leading, multidisciplinary scientific journals Nature, Science and PNAS in 2019. A second (disciplinary) sample was selected to explore publication and co-authorship rates of ECRs who routinely publish in discipline-specific journals. This sample was also to compare whether trends observed in the publication rates of leading scientists, are also seen in a cohort of researchers exclusively publishing in more specialised journals – perhaps a more familiar experience for most ECRs. The disciplinary sample included all authors who in 2019 published in any journal that the authors of this article had published in at least twice, including a wide range of ecology, interdisciplinary landscape, and environmental psychology journals (n=21, Table 1).\n\nThe ECR publication history of these two author samples were collected using the Scopus API, accessed using the rscopus package in R v3.6 in January 2020. A list of all 2019 publications with a Scopus type of ‘Article’ or ‘Review’ (and ‘Letter’ in Nature) were retrieved for each journal of interest using the scopus_search function based on the ISSN of each publication.45 All authors for each article were retrieved.\n\nThere is no consensus on the definition of ‘early career’. Previous studies have defined ECRs as researchers within five to twelve years of their first year of publishing.16,25 In a study of research productivity by career stage, ECRs had an average of 4.5 year’s experience post-PhD, compared with 14.1 years for mid-career faculty members.26 In this study we defined ‘early career’ as the first ten years of a career to encompass a broad range of definitions – subsets of the data presented here can be applied to shorter definitions of ECR career length.\n\n‘Career age’ or ‘academic age’ are often defined as the number of years since first publication,11,13 or years since PhD.27 However, consistent with other studies,28 we observed that many researchers in our author cohorts had gaps in their early years of publishing, possibly due to career gaps [e.g. due to parental leave, illness or caring responsibilities] or breaks between completing honours or masters degrees and beginning PhD studies along with the increasing prevalence of publishing before completing a PhD.22 As early career publishing rates are sensitive to these gaps, we controlled for them by calculating the career age as the number of ‘active’ years of publishing, i.e., excluding years with no publications. While recognising that continuous publishing is an important predictor of academic excellence,28 excluding these career gaps better reflects the publishing experience of most ECRs.\n\nThe early career publication history (i.e. the first ten active years) of n=55,332 authors for the multidisciplinary sample and n=85,793 authors for the disciplinary sample was retrieved using the author_retrieval function of the rscopus package. Details of articles including publication date, publication title, and co-authors, and author details including institution, city, and country were extracted. Erratum and corrigendum were removed from the analyses. For each published article (n= 1,195,246 articles for the multidisciplinary sample and n= 1,597,069 articles for the disciplinary sample), the author position, and number of co-authors were determined.\n\nFor each active year of publication for each author, the number of publications, number of first author publications, total number of unique co-authors, and the average number of co-authors per publication was calculated. Adopting the approach of Calver et al., we drew on ecological statistics, using the Jaccard index to calculate co-author dissimilarity across publications using the vegdist function in the vegan package in R.29 This measure of dissimilarity will approach 1 when co-authors vary across publications, and approach 0 when co-authors are similar across all publications, in any given year.\n\nA generalised linear model using a Poisson distribution was used to predict publication rates (total number of publications, and number of first author publications) and a log model was used to predict co-authorship rates (average co-authors per publication) and a Gaussian distribution to predict co-author dissimilarity across publications, from the first year of publication and career age.\n\nIn the context of increasing concern over the focus on quantity over quality in academic publishing and research evaluation,3 we explored alternative metrics that could provide benchmarks for ECR publication rates using the multidisciplinary cohort. We calculated the median and mean number of publications for all multidisciplinary authors for each active career year, and the median number of first author publications of all multidisciplinary authors across all active years. We also provide comparison publication rates for authors from different co-authorship environments: authors in the top quartile of average number of co-authors over the last decade and for authors from the bottom quartile of average number of co-authors over the last decade.\n\nBased on these data, we determined measures of publishing success that limit publication expectations using three metrics: did the author publish at least the: 1) median, 2) mean number of publications for their career age in any given year, and 3) did the author publish at least the median number of first-author publications in any given year? A set of logistic models was developed to predict the probability of meeting these metrics from the average number of co-authors per publication (log transformed), and the dissimilarity of authors across publications (as measured by the Jaccard index).\n\n\nResults\n\nECR publication rates have increased over the last 50 years, particularly in our disciplinary sample which is now publishing at higher rates than authors in Nature, Science and PNAS. Multidisciplinary ECR publication rates have increased 46% between 1970 and 2019 (Figure 1A). Current ECRs are publishing an average of 3.5 publications/year after five active years, compared with 2.4 publications/year after five years for ECRs in 1970 (Figure 1A). This increase is dwarfed by the increases in publication rates that occur over the first ten years of a career. Current ECRs publication rates increase threefold over the first ten years of publishing, from 2.1 publications/year in the first year of publishing to 6.6 publications/year in the tenth year of publishing. There were contrasting findings for first author publication rates, which have declined 60% over the last 50 years. After five active years of publishing, authors in 1970 were publishing 1.5 first author publications per year, while in 2019 this figure was 0.6 first author publications per year (Figure 1B). There was a smaller increase in first author publication rates over the first ten years of a career, which increase 31% between the first and tenth active year of publishing for current ECRs (Figure 1D).\n\nThe increase in publication rates over time was much more dramatic in our disciplinary sample, increasing 105% between 1970 and 2019, from rates that were substantially lower than the multidisciplinary sample to rates that are now higher. After five active years of publishing, rates had increased from 1.7 publications/year in 1970 to 3.6 publications/year in 2019 and from 3.4 to 7.1 publications/year after ten years (Figure 1D). There was a smaller decline of 28% in first author publications per year, from 1.3 in 1970 to 0.9 in 2019 after ten years of publishing.\n\nThere were substantial increases in average co-authorship rates over the last 50 years, particularly amongst multidisciplinary ECRs (Figure 2A), who are now publishing with 572% more co-authors per publication compared with 50 years ago. The average number of co-authors per publication in the tenth career year increased from an average of 2.9 authors per publication in 1970 to 16.6 in 2019. In our disciplinary sample, this increased 230% from 2.9 co-authors per publication in 1970 to 9.5 co-authors per publication in 2019 in the tenth career year. There were also increases in co-authorship rates over the first ten years of publishing of 61% in the multidisciplinary sample and 49% in the disciplinary sample. Dissimilarity of authors across publications has increased slightly from 1970 to 2019 in the multidisciplinary and disciplinary samples. However, dissimilarity increased substantially over the first ten years of a career in both samples (Figure 2B and Figure 2D).\n\nOur new measure of active year (years since first publication, excluding years with no publications) was similar to career age (years since first publication; Figure 3A). One quarter of authors had no publication gaps in their first ten years of publishing, while 58% of authors had three or fewer years with no publications (Figure 3B). While the differences between active year and career age may be relatively small, active years should more accurately describe the opportunity of ECRs who have had career interruptions or a break between publishing from honours or master’s studies, than career age or number of years since PhD completion.\n\nOur selected benchmarks for evaluation were the career-age controlled median and mean publication rates of 55,332 authors publishing in Nature, Science and PNAS in 2019 (Table 2, Figure 4), and publishing at least the median number of first-author publications per year relative to their cohort. Median publication rates increased from one publication/year in the first active year of publishing, to two in the fifth year of publishing to four in the tenth year of publishing (Table 2). Mean publication rates increased due to a number of authors with substantially higher publication rates (as seen by the outliers in Figure 4), from 1.6 publications/year in the first active year of publishing, to 3.3 in the fifth year of publishing to 5.2 in the tenth year of publishing (Table 2). Median and mean publication rates were somewhat higher (34%) for authors with many co-authors, and slightly lower (13%) for authors with few co-authors. The median first author publication rate was one first author publication/year in the first ten years of publishing. In any given year 71% of multidisciplinary authors met the median publication rate benchmark, 33% met the mean publication rate benchmark, and 53% met the first author publication benchmark.\n\nRates for authors with many co-authors (top quartile>13.1 average co-authors) and few co-authors (bottom quartile<5 average co-authors) are shown for comparison.\n\nMedian values are shown as thick horizontal lines, mean values as solid circles, and the box shows the interquartile range.\n\nThe likelihood of meeting these benchmarks was higher for ECRs whose co-authors vary across publications each year (Figure 5, Table 3) as measured by co-author dissimilarity (Jaccard index). This is not surprising given that publishing with different authors necessarily means publishing more. Increasing the number of co-authors per publication also had positive effect (although smaller than author dissimilarity) on the likelihood of achieving median and mean publication rates (Figure 5A, C, Table 3). However, publishing with a greater number of co-authors per publication greatly decreased the probability of having a first-author publications (Figure 5F, Table 3).\n\n95% confidence intervals are shown.\n\n\nDiscussion\n\nCurrent ECRs have higher rates of publication than ECRs from earlier decades. This is true for scientists publishing in leading multidisciplinary journals, and much more so for a cohort of ECRs publishing in disciplinary journals spanning ecology, interdisciplinary landscape, and environmental psychology. In the context of these findings, and heeding the call to “abandon paper counting”,3 we present three evidence-based benchmarks of research performance for ECRs: the median and mean yearly publication rates, and median yearly first-author publication rates, relative to their disciplinary cohort. The best predictor of meeting these publication benchmarks was publishing with co-authors that vary from publication to publication. Perhaps surprisingly, having more co-authors per publication was negatively related to meeting the first-authorship benchmark.\n\nThe acceleration in publication rates observed in our study is unlikely to be sustainable. While there are undoubtedly ECRs who publish at the higher rates observed over the last decade while maintaining research quality and impact (and quality of life), this is unlikely to be true for many researchers. In order to develop a competitive track record, ECRs are often advised to work long hours, avoid activities that aren’t directly linked to career advancement,30 and write without the distractions of work colleagues, friends and families.31,32 These effects may be compounded by short-term contracts which demand continual output at the expense of “human and social capital accumulation”, and can lead to career termination due to unavoidable fluctuations in output.33 An unbalanced focus on publication rates risks compromising other aspects of scientific endeavour and academic development, and it may lead to declines in research quality and impact, compromise work-life balance and mental health, or reduce diversity as disadvantaged researchers slip from the system.18,30,34\n\nInterestingly, rates of first authorship are decreasing, particularly in the multidisciplinary author group. This seems a natural corollary to the even greater increases observed in co-authorship rates over the last 50 years, as has previously been well demonstrated for established researchers,35 and may also be partly offset by increases in joint first-authorship publications.36 While the average number of co-authors increases slightly over the first ten publishing years of an academic career, there has been a substantial increase over the last 50 years for all ECRs. This demonstrates a substantial change in publishing patterns, away from individual and small group collaborations to larger team collaborations. Relatively stable patterns in the dissimilarity of co-authors across publications highlight that this shift is to relatively stable larger teams of authors rather than larger individual networks of co-authors.\n\nConcerningly, the real size of the observed acceleration in publication rates is likely to be greater than described here. Our data is likely to be oversampling the most successful established scientists, as ECR’s from earlier decades who ceased publishing before 2019 are not included. This is consistent with previous research which has identified the attrition of less-productive researchers as a factor in assessing publication rates over time.27\n\nThere is a clear need for evaluation strategies that are tailored to variations in publication rates over the early years of a career, and do not further encourage unsustainable rates of publication – that avoid assuming ever increasing quantities of publications are better.18 We have proposed a suite of evidence-based benchmarks with which to set ECR publishing expectations, and to evaluate ECR publication performance. These benchmarks incorporate career age, ensuring that they more appropriately and fairly assess performance of those early in their career. A suite of metrics allows for a more comprehensive view of research productivity (e.g., volume, collaboration, and research leadership), while still permitting ‘outstanding’ performance to be identified. For example, there is a substantial difference between median and mean publication rates as a result of enormous variation in the output of individual researchers, with some ECRs in this sample publishing more than 100 papers in some years. This difference is potentially useful for evaluating ECR productivity as the proportion of researchers meeting mean publication rate benchmarks was half the rate of those meeting median publication rates. This allows some discrimination between ECRs based on publication productivity, while still limiting the expectations on the numbers of publications used in evaluations. ECRs and institutions could use these to set expectations for sensible rates of publishing or to choose the number of publications to use in research evaluation.3\n\nCare must be taken in the use of this evidence base. It does not control for part-time work and the fraction of employment devoted research. While it does control for career gaps spanning calendar years, it does not control for shorter career gaps or periods of reduced activity due to parenting, caring responsibilities, health and other pressures that can substantially affect research productivity. It is likely that there is some variation in publication rates across different research areas, and future research may develop subject-specific benchmarks. Further research could explore the role of different kinds of publications such as books that are important in some disciplines.26\n\nThe finding that publishing with dissimilar authors across publications is a better predictor of meeting these benchmarks than large numbers of co-authors per paper, is consistent with studies of academic success in established researchers that highlight the benefits of collaboration, and the pitfalls of over-reliance on publications with many authors.24,33,37,38 Previous studies have shown that publishing with a moderate number of co-authors is associated with higher publication and citation rates.39 Further afield, international collaboration is associated with increased publication productivity.40,41\n\nThe strategy of developing a broad and varied network of co-authors may also lead to a range of other benefits such as grant writing opportunities, exposure to diverse views and development of professional networks, and consequently job opportunities. Research suggests that opportunities for networking and collaboration can lead to an increased perception of quality of a research environment and improved publication rates for ECRs.42 More broadly, increased networking can lead to perceived and actual benefits in the workplace,43 through mechanisms such as increased access to information, resources and mentoring.44\n\n\nStudy limitations\n\nThe multidisciplinary author sample used here have all published in top journals that most researchers do not publish in. Thus, basing benchmarks on the ECR track record of these authors may not be fair to all ECRs. Nonetheless, the comparison with a disciplinary author sample that has largely converged with the publishing rates of these authors provides some confidence that the benchmarks will be valid, at least in some science disciplines. Further research could help to better understand which disciplines these benchmarks are indeed valid for, and which disciplines require alternative benchmarks (or for which publishing benchmarks may not be appropriate).\n\nWhile Scopus historical data is likely to be limited and may not include all publications of ECRs from the 1970s and 1980s, this limitation is balanced to an extent by our sample only including authors who continue to publish in top journals in 2019– and this group is likely to have higher publication rates than the average ECR from the 1970s and 1980s.\n\n\nConclusion\n\nThere is growing concern about the effects of the ‘publish or perish’ mentality on the career prospects and wellbeing of young researchers.18,20 In response, some authors are arguing that the number of publications included in research evaluations should be limited.3 Our findings show that current early career researchers are indeed publishing at an accelerated rate compared with their peers from previous decades. This is more pronounced for our disciplinary peers compared to authors in leading multidisciplinary journals. This work is a first step in introducing an evidence base to set publishing expectations and support the current debate around limiting publication rates and the number of publications used in research evaluations for ECRs. Reducing the pressure to always be publishing more, may allow ECRs to focus on the broader societal and institutional responsibilities that come with a career as a researcher, while still demonstrating research excellence. This may encourage ECRs to invest time in other endeavours leading to personal, academic, and societal benefits, such as grant applications, student supervision, collaboration, teaching, communication, outreach, industry engagement and academic service. These are critical pillars of an academic career. Framed in this way, developing collaborative networks with other researchers and research partners becomes a useful strategy for success rather than a distraction from writing more publications. These findings do not diminish the need for qualitative, place and mission specific assessments of research performance,2 but instead provide a suite of metrics that can more reasonably guide and evaluate the publication performance of early career researchers.\n\n\nData availability\n\nHarvard Dataverse: ECR publication metrics https://doi.org/10.7910/DVN/SM9KMI45\n\nThis project contains the following underlying data:\n\n• pubs_year-metadata-Jan2021.tab\n\n• pubs_year-multi.csv\n\n• pubs_year-us.csv\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to thank Colin Simpfendorfer and Helene Marsh for comments on an earlier version of this manuscript.\n\n\nReferences\n\nChapman CA, Bicca-Marques JC, Calvignac-Spencer S, et al.: Games academics play and their consequences: How authorship, h-index and journal impact factors are shaping the future of academia. 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Publisher Full Text" }	[ { "id": "124516", "date": "07 Mar 2022", "name": "Gerben ter Riet", "expertise": [ "Reviewer Expertise Clinical epidemiology", "meta-research", "research integrity", "publication bias" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors set out to provide the debate(s) on academic (over)production, slow(er) science and better work-life balance with empirical evidence, focusing on early career researchers (ECRs). In particular, they produced reference values (benchmarks) for what may be the expected number of papers an average ECR publishes, stratified by his/her year after career start. These benchmarks, according to the authors, may serve in research evaluations of ECRs.\nIn addition, they show how these production counts depend on the number and variability of co-authors on papers. Finally, they show by how much the number of publications ECRs produce has changed (increased) over the last 5 decades.\nTo me paper would gain much focus if it dealt with objective 2 only:\n\nDeveloping evidence-based benchmarks for ECR publication rates as careers develop.\nThat is a rich topic and worthwhile goal in itself.\n\nMy personal opinion is that: I sympathize with the idea that science should slow down and focus on quality more. In the same vein, I worry that the benchmarks the authors produce, if valid, might also be used against ECRs by employers or funders that require an ECR to be at least above the 75th quantile. So, a more radical approach in research evaluations is that taken by the Dutch scientific authorities where they do away with counts altogether. (I am not a co-author of these guidelines). https://www.universiteitenvannederland.nl/files/documenten/Domeinen/Onderzoek/SEP_2021-2027.pdf (in English).\nIn their balanced discussion, the authors acknowledge their work as a first step and also emphasize that: \"Further research could help to better understand which disciplines these benchmarks are indeed valid for, and which disciplines require alternative benchmarks (or for which publishing benchmarks may not be appropriate).\"\nPatrick Royston (1995)1 has developed a method to derive benchmarks (reference values) in which covariables can be incorporated. I wonder if the authors might benefit from Royston’s methodology and derive model-based benchmarks including 95% confidence intervals around the best fitting line as well as around the lines that form the x-th (e.g. 90th) centile ranges.\nBelow I follow the various paragraphs in the paper:\nIntroduction, paragraph 1\nWhat is exactly meant by “managing academic expectations”. Reference 1 does not mention this term as far as I could see. This should be clarified.\n\nThe authors switch from a ‘focus’ to an ‘unbalanced focus’ on publication counts, but I think that it is important that the authors distinguish these, because some (limited) usage of metrics is quite different – and may be defensible – from evaluations in which they are central.\n\nThe authors should state far more clearly what they see as quality and impact (or valid indicators of those)? I think that publication counts is a far more primitive measure than citations (not self-citations!) and journal impact factor and should not be in the same category. The author seem to acknowledge this in paragraph 2 where they use citation rates as a measure of quality that is not all bad, saying: “It is a poor predictor of later research ‘quality’ as measured by indicators such as citation rates. 19 ”, but its condemnation here and approval in paragraph 2, to me, is inconsistent.\n\nThe authors worry that evaluations may be reduced to competitions of researcher status. But to me, researcher status is something inherent to any evaluation system? Whatever systems for evaluation we build, each will produce its champions. In ideal (?) circumstances, we may view evaluations as mere starting points for a discussion with others (your boss, funders, ..) on what is needed for ECRs or institutes to become (even) better, not as justifications to stop funding or firing someone. But is that realistic?\nIntroduction, paragraph 2\nI wonder if the h-index is also a bad measure if it were used to compare researchers within, say, a 3-year working career range, a career-age stratified h-index? If this cannot be realistically done, the authors should argue along these lines.\n\nPublishing with top scientists can be an asset of an ECR, but will usually occur via the network of one of his/her seniors, which may be an indicator of “working at a more prestigious institution”. But, anyhow, having access to such people may be an, albeit, indirect indicator of an ECR’s own quality, since prestigious institutions may apply heavier hurdles to become a member.\n\nThe authors should distinguish between the measurement of an ECR’s success today and previously measured predictors (future) of having success. A combination of both may convey additional information, since e.g. having no success today while having had a favorable prediction score in the past may indicate that despite an optimal academic environment one did not succeed. The reverse of course is an excellent ECR who has been working in a modest environment and is nevertheless successful today, which may highlight exceptional qualities (or luck).\n\n“In the context of uncapped publication expectations”. I think that the authors should distinguish between personal evaluations within research institutes or when seeking funds and (group or team) evaluations of institutes of which an ECR may be a member. In The Netherlands, the SEP intended for use between 2021 and 2027 for research institutes de-emphasizes count outcomes and h-indices (seemingly ignoring career duration stratified h-indices) and has moved toward adopting group narratives, although it still includes research grants awarded to individuals, grants awarded to major collaborative research projects and prizes awarded to individuals or collaborative research projects: https://www.universiteitenvannederland.nl/files/documenten/Domeinen/Onderzoek/SEP_2021-2027.pdf (in English). I struggled to grasp what the customs are in Australia, and concluded that institutes have the freedom to take a sample of their output and present for formal evaluation.\nIntroduction, paragraph 3\nThe authors write “There is currently little empirical data to make sense of these debates for ECRs..” I think that some of the confusion (or lack of focus) in the introduction stems from not being explicit on which debate the authors want to focus on exactly. It seems they want to derive reference (‘normal) ’values (“career stage-specific benchmarks”, see study objectives) for publication counts (like labs have reference values for blood constituents), but how does this match with their critique of such counts? The authors should clarify their stance on this.\nIntroduction, paragraph 4\nThe authors state that “A research evaluation framework that is specific to the early years of a career is needed. This should be based on an understanding of how ECR publication and co-authorship rates have changed through time, how they progress during a career, and what strategies best support research ‘success’.” Let’s suppose that such a framework is indeed needed, why should it be based on understanding publication rates through time or how they progress over careers? The authors should provide compelling arguments for their assertions.\n\nI am not convinced that their research questions 3 make sense. Can the authors explain why estimating the effect of such strategies is better than incorporating the strategies into the benchmarks themselves which to me seems a more logical approach?\n\nGiven that citation rates and journal impact factors may be somewhat more refined measures than pure publication counts, wouldn’t it be more valuable to develop benchmarks for the former two indicators? Please comment.\n\nHow were trends, if any, in part-time working factors accounted for? The authors mention this as a limitation in their discussion on the benchmarks, but do not in their discussion of the trends over time.\n\nThe study may suffer from selection bias (survivorship bias to be specific) (and the authors acknowledge this, as a limitation, although not using this term): first, ECRs who dropped out because they couldn't accept the current environment of hyper-competition and authors who did not publish anything in 2019 were not included in the study sample.\n\nRelated to the point above, the authors state in their Discussion that: \"Concerningly, the real size of the observed acceleration in publication rates is likely to be greater than described here. Our data is likely to be oversampling the most successful established scientists, as ECR’s from earlier decades who ceased publishing before 2019 are not included.\" I drew the exact opposite conclusion and think that the observed (real!) acceleration is smaller than described here, because those who did (and do) not survive in the current hypercompetitive environment have been left out. The authors should clarify their line of thought.\n\nHow might the authors account for delays in publishing between journals or fields (humanities, math)? If not, they should add this as a limitation.\n\nDid the authors cut out time gaps with zero publication using calendar years or years since last date of publication and did they use dates of submission or dates of publication? Please clarify.\n\nWere retractions taken into account or are these numbers negligible.\nResults section.\nIn figure 5, the 95% confidence intervals are missing.\n\nData and syntaxes.\nPlease add the code to extract the data from Scopus and the exact R code used for extractions.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "134547", "date": "14 Apr 2022", "name": "Laurel L Haak", "expertise": [ "Reviewer Expertise Research workforce policy", "research infrastructure", "collaboration", "team science", "author disambiguation", "research evaluation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 examines options for measuring outputs of ECRs, to get away from over-reliance on paper counting in researcher evaluation. It uses publication data and normalizes mean and median publication volumes based on active years.\n\nThe paper could be further improved by addressing the following points:\nWhile the paper presents well-founded proposals for assessing ECR output, it still relies fully on publication data.\n\nThe authors do not address possible confounds with author disambiguation issues in the methods or results section.\n\nThe data made available do not appear to include R scripts or author data.\n\nThe introduction and conclusion sections could have referenced more of the recent literature on team science and collaborative research trends.\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? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-95
https://f1000research.com/articles/11-92/v1	25 Jan 22	{ "type": "Software Tool Article", "title": "The software for interactive evaluation of mass spectrometric imaging heterogeneity", "authors": [ "Evgeny Zhvansky", "Ekaterina Zhdanova", "Maxim Belenikin", "Maria Shamraeva", "Sergei Silkin", "Konstantin Bocharov", "Anatoly A. Sorokin", "Ekaterina Zhdanova", "Maxim Belenikin", "Maria Shamraeva", "Sergei Silkin", "Konstantin Bocharov", "Anatoly A. Sorokin" ], "abstract": "Mass spectrometry imaging is a promising tool complement to the histology study for evaluation of presence of different tissue types in the sample. To make this method faster, more accurate and precise we have presented earlier the cosine similarity measure maps (CSMM). The method provides the spatial distribution of cosine similarity measure metrics between chosen MSI pixel and the rest of the image. In cases when samples under test are heterogeneous and not guaranteed to have clear clusters with distinct borders, it is interesting to analyze the heterogeneity, area borders and their sensitivity to reference CSMM pixel selection. Here we present the software for interactive building of CSMM for different parameters, their visual analysis and saving such CSMM in publication-ready quality without additional programming. Source code, example datasets, binaries, and other information are available at https://github.com/EvgenyZhvansky/Interactive_CSMM.", "keywords": [ "mass spectrometric imaging", "interactive analysis", "clustering", "labeling" ], "content": "Introduction\n\nMass spectrometry imaging (MSI) is a technique of building a map of the spatial distribution of molecular features across the tissue of interest without pretreatment.1–4 This technique is a promising complement to the gold standard of tissue analysis – histology study that is a time-consuming, labor-intensive, and sometimes subjective method.\n\nEach pixel of raw MSI-map is a mass spectrum of the corresponding location in the sample. This multidimensional map is almost impossible to interpret by naked eyes and has to be converted to a simpler representation for better visualization and usability.5–12\n\nIn our previous work, we introduced13 a fast, precise, and accurate imaging tool based on the interactive building of the cosine similarity measure maps (CSMM) between the reference pixel and the rest of the image. Introduced technique well suited for visual estimation of presence, location, and level of heterogeneity of homogeneous regions in the image. It also allows extraction of the region reference mass spectra, and evaluation of the influence of the reference pixels on the distribution of similarity characteristics on the map.\n\nHere we present a user-friendly interface for building and analysis of CSMMs of MSI data.\n\n\nMethods\n\nHere we introduce Interactive CSMM, a MATLAB app, which provides an intuitive graphical interface for interactive evaluation of mass spectrometric imaging heterogeneity. An example of the interface is shown in Figure 1. Interactive CSMM was created in MATLAB R2019b. We also introduce the Python script for converting standard raw imzML file format to mat-file, which is required by the Interactive CSMM. The script currently depends on the following libraries: numpy, psutil, pyimzML and scipy.\n\nOn the right panel on the top left CSMM is located, on the top right the result of CSMM median smoothing is displayed, on the bottom left is a map of the boundaries of homogeneous regions, and the mass spectrum of the reference pixel is shown on the bottom right.\n\nThe Interactive CSMM can be launched locally from any computer with MATLAB (R2019b or higher; lower versions also might work properly) installed. Installation and launching instructions are also available. All interfaces and plots of Interactive CSMM are highly interactive, allowing users to visualize data in real-time with interactive selection reference mass spectrum, as well as store the results of the analysis.\n\nThe program 1) allows the user to interactively select the reference pixel, specify the mass range and other parameters for data binning 2) calculates the CSMM of all imaging data with respect to the selected pixel 3) label homogeneous areas and save the assigned area number, coordinates of the reference pixel and the name of the CSMM image file built on this pixel to a text file 4) save publication-ready images of CSMM with specified resolution.\n\nTo improve the interpretability of the image and clean up the pixelation, outliers, and measurement artifacts the program provides the smoothed version of CSMM, the visual map of the boundaries of homogeneous zones,13 and the mass spectrum of the reference pixel.\n\nThe presence in the spectra ions distributed over the tissue equally could cause blurring of the picture. So, we provide users with options to specify ranges is m/z which are reflecting the tissue heterogeneity, and compare positions and shape of homogeneity regions obtained in different m/z ranges.\n\nAnother effect that complicates the interpretation of MS images is the presence of transition zones due to gradual changes in the ion’s intensity within such zones. In our method, due to the building of the CSMM over the mass range, the transition zones become clearly visible because they include the peaks of both boundary zones. That effect is more difficult to achieve with standard imaging approaches (Supplementary Materials of the method describing article13), which consider the distribution of individual ions.\n\nThe additional benefit of our method is that there is no data preprocessing is required other than binning. The parameters of binding can be changed online. No alignment is required as well, it can be replaced with a larger binning. It could be also shown that the method works well with non-normalized data and there is no need for baseline correction.\n\nCSMM allows you to define zones of least and greatest similarity. It does not automatically divide the measured map into zones. But you can find areas with similar spectra by varying the reference pixel. By changing the other parameters (to a greater extent, the mass range), it is possible to optimize the CSMM color map and improve the visualization of heterogeneity of the measured sample. The smooth changes in heterogeneity can be observed as smooth color changes on the CSMM.\n\nWe tested this method on different data sources14 (measured with different ion sources: MALDI-imaging, DESI-imaging; and different mass analyzers TOF MS, Orbitrap, ICR MS) (Supplementary Materials of the method describing article13). Our article presents CSMMs for colorectal adenocarcinoma data.\n\nPreprocessing steps and descriptions of operations are presented in the manual.\n\n\nConclusion\n\nWe presented software that allows users to quickly evaluate the presence and structure of heterogeneous areas in the sample, and manually make a dataset of feature spectrum for the homogeneous zones. By varying the reference spectrum and the mass range used in the construction of the CSMM, it is possible to understand which mass ranges most reflect the heterogeneity and make the boundaries between the zones more contrasting. A more detailed discussion is presented in the method describing article.13\n\n\nData availability\n\nGigaDB: Supporting materials for “Benchmark datasets for 3D MALDI- and DESI-Imaging Mass Spectrometry”, http://dx.doi.org/10.5524/100131.14\n\nThis project contains the following underlying data:\n\n- 3DMouseKidney.ibd\n\n- 3DMouseKidney.imzML\n\n- 3D_Mouse_Pancreas.ibd\n\n- 3D_Mouse_Pancreas.imzML\n\n- 3D_OSCC.ibd\n\n- 3D_OSCC.imzML\n\n- ColAd_Individual.zip\n\n- Colorectal_Adenocarcinoma.h5\n\n- Microbe_Interaction_3D_Timecourse_LP.ibd\n\n- Microbe_Interaction_3D_Timecourse_LP.imzML\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nData are also available at Metabolights: MTBLS176: Benchmark datasets for 3D MALDI- and DESI-Imaging Mass Spectrometry.\n\n\nSoftware availability\n\nSource code available from: https://github.com/EvgenyZhvansky/Interactive_CSMM/.\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.5776541.\n\nLicense: Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor contributions\n\nConceptualization A.S., E.S.Z.; methodology A.S., E.S.Z.; software E.S.Z.; investigation M.B., E.V.Z; writing — original draft preparation E.S.Z., E.V.Z., M.S.; writing — review and editing E.S.Z., E.V.Z., A.S., K.B.; visualization E.S.Z.; supervision A.S.; project administration A.S.; funding acquisition A.S.; formal Analysis E.S.Z.; data curation M.B., M.S., S.S., K.B.; validation E.V.Z., M.B.; resources M.B., E.V.Z. All authors have read and agreed to the current version of the manuscript.", "appendix": "Acknowledgments\n\nThe research used the equipment of Shared Research Facilities of N.N. Semenov Federal Research Center for Chemical Physics of the Russian Academy of Sciences.\n\n\nReferences\n\nMcDonnell LA, Römpp A, Balluff B, et al.: Discussion point: reporting guidelines for mass spectrometry imaging. Anal. Bioanal. Chem. 2015 Mar; 407(8): 2035–2045. PubMed Abstract \| Publisher Full Text\n\nHoratz K, Giampà M, Karpov Y, et al.: Conjugated Polymers as a New Class of Dual-Mode Matrices for MALDI Mass Spectrometry and Imaging. J. Am. Chem. Soc. 2018 Sep 12; 140(36): 11416–11423. PubMed Abstract \| Publisher Full Text\n\nBuchberger AR, DeLaney K, Johnson J, et al.: Mass spectrometry imaging: A review of emerging advancements and future insights. Anal. Chem. 2018 Jan 2; 90(1): 240–265. PubMed Abstract \| Publisher Full Text\n\nHanrieder J, Gerber L, Persson Sandelius Å, et al.: High resolution metabolite imaging in the hippocampus following neonatal exposure to the environmental toxin BMAA using ToF-SIMS. ACS Chem. Neurosci. 2014 Jul 16; 5(7): 568–575. PubMed Abstract \| Publisher Full Text\n\nWüllems K, Kölling J, Bednarz H, et al.: Detection and visualization of communities in mass spectrometry imaging data. BMC Bioinform. 2019 Jun 4; 20(1): 303. PubMed Abstract \| Publisher Full Text\n\nKaddi CD, Parry RM, Wang MD: Multivariate hypergeometric similarity measure. IEEE/ACM Trans. Comput. Biol. Bioinform. 2013 Dec; 10(6): 1505–1516. PubMed Abstract \| Publisher Full Text\n\nAlexandrov T, Kobarg JH: Efficient spatial segmentation of large imaging mass spectrometry datasets with spatially aware clustering. Bioinformatics. 2011 Jul 1; 27(13): i230–i238. PubMed Abstract \| Publisher Full Text\n\nWüllems K, Zurowietz A, Zurowietz M, et al.: Fast visual exploration of mass spectrometry images with interactive dynamic spectral similarity pseudocoloring. Sci. Rep. 2021 Feb 25; 11(1): 4606. PubMed Abstract \| Publisher Full Text\n\nRace AM, Palmer AD, Dexter A, et al.: Spectralanalysis: software for the masses. Anal. Chem. 2016 Oct 4; 88(19): 9451–9458. PubMed Abstract \| Publisher Full Text\n\nBokhart MT, Nazari M, Garrard KP, et al.: MSiReader v1.0: Evolving Open-Source Mass Spectrometry Imaging Software for Targeted and Untargeted Analyses. J. Am. Soc. Mass Spectrom. 2018 Jan; 29(1): 8–16. PubMed Abstract \| Publisher Full Text\n\nKlinkert I, Chughtai K, Ellis SR, et al.: Methods for full resolution data exploration and visualization for large 2D and 3D mass spectrometry imaging datasets. Int. J. Mass Spectrom. 2014 Apr; 362: 40–47. Publisher Full Text\n\nRàfols P, Torres S, Ramírez N, et al.: rMSI: an R package for MS imaging data handling and visualization. Bioinformatics. 2017 Aug 1; 33(15): 2427–2428. PubMed Abstract \| Publisher Full Text\n\nZhvansky ES, Ivanov DG, Sorokin AA, et al.: Interactive Estimation of Heterogeneity from Mass Spectrometry Imaging. Anal. Chem. 2021 Mar 2; 93(8): 3706–3709. PubMed Abstract \| Publisher Full Text\n\nOetjen J, Veselkov K, Watrous J, et al.: Supporting materials for “Benchmark datasets for 3D MALDI- and DESI-Imaging Mass Spectrometry.”. GigaScience Database. 2015; 4: 20. PubMed Abstract \| Publisher Full Text" }	[ { "id": "160344", "date": "10 Feb 2023", "name": "Jean-Nicolas Audinot", "expertise": [ "Reviewer Expertise Mass Spectrometry 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\nThis publication is about the description of a software that allows users to evaluate the presence and structure of heterogeneous areas in the (biological) sample. By varying the reference spectrum and the mass range used, the software allow the construction of the the cosine similarity measure maps (CSMM). The objective of this work (data treatment) is to understand which mass ranges most reflect the heterogeneity. This work was already presented in a previous publication1.\nPoint 1: The originality with the previous publication1 is not obvious. I found more information in the previous publication, like the definition of the CSMM. Maybe wrongly, due to lack of time, as I couldn't test all the (new) option, I didn't see any difference with the previous version\n\nPoint 2: The expected data is not very clear. the manual provided with the program is really poor. It should at least be enriched with many examples, and especially with processed data. It's a pity, we'll have to wait for publications with this software to really know its potential\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-92
https://f1000research.com/articles/11-91/v1	25 Jan 22	{ "type": "Review", "title": "Contaminants in the cow's milk we consume? Pasteurization and other technologies in the elimination of contaminants", "authors": [ "Micaela Belen Calahorrano-Moreno", "Jonathan Jerry Ordoñez-Bailon", "Ricardo José Baquerizo-Crespo", "Alex Alberto Dueñas-Rivadeneira", "Maria Conceição B. S. M. Montenegro", "Joan Manuel Rodríguez-Díaz", "Micaela Belen Calahorrano-Moreno", "Jonathan Jerry Ordoñez-Bailon", "Ricardo José Baquerizo-Crespo", "Alex Alberto Dueñas-Rivadeneira", "Maria Conceição B. S. M. Montenegro" ], "abstract": "Cow's milk is currently the most consumed product worldwide. However, due to various direct and indirect contamination sources, different chemical and microbiological contaminants have been found in cow's milk. This review details the main contaminants found in cow's milk, referring to the sources of contamination and their impact on human health. A comparative approach highlights the poor efficacy and effects of the pasteurization process with other methods used in the treatment of cow's milk. Despite pasteurization and related techniques being the most widely applied to date, they have not demonstrated efficacy in eliminating contaminants. New technologies have appeared as alternative treatments to pasteurization. However, in addition to causing physicochemical changes in the raw material, their efficacy is not total in eliminating chemical contaminants, suggesting the need for new research to find a solution that contributes to improving food safety.", "keywords": [ "human health", "chemical contaminant", "microbiological contaminant", "alternative", "technology", "food safety" ], "content": "1. Introduction\n\nMilk is a fluid secreted by the female of the mammalian species and fulfills the nutritional requirements of the neonate, for instance: (i) the energetic part (provided by lipids, lactose, and in excess by proteins), essential amino acids, and (ii) amino groups necessary for the biosynthesis of non-essential amino acids (provided by proteins), essential fatty acids, vitamins, inorganic elements, and water.1\n\nGlobal milk production has increased by about 20% in the last decade, from 694 million tons in 20082 to 843 million tons in 2018.3 As a result, bovine milk is the most consumed food product representing about 48% of the total milk consumed globally, the European Union (EU), Australia, and New Zealand being the most important producers, followed by the United States and India.4\n\nCollection and processing expose milk to different contaminants, mainly pesticide residues, metals, mycotoxins, hormones, and others reaching the cow through feeding or drug administration by producers.5 Thus, milk can contain hazardous materials, of either biological or chemical origin.\n\nAlthough pasteurization has been an efficient antimicrobial method and has contributed to reducing many diseases, several infectious episodes associated with pasteurized milk have continued to occur, mainly when raw milk has an exaggerated population of microorganisms that increase the margin of survival and by post pasteurization contamination.6 The biggest problem of pathogens in pasteurized milk is that they persist without causing any organoleptic alteration, increasing sanitary risk since the consumer cannot suspect their presence, showing that pasteurization has some drawbacks in treating pathogens.7\n\nAs population and industrial growth increased, new contaminants appeared, and with this, contamination of cow's milk also increased not only by compounds of biological origin but also by compounds of chemical origin, as mentioned above.8 However, pasteurization has remained the only established treatment, even though it is only effective for eliminating most biological and non-chemical compounds.9 In contrast, the literature mentions very few alternative treatments to treat chemical contaminants in cow's milk, leading to a critical analysis of their application to ensure sufficient quality in the milk consumed. Given this evidence, the bibliographic review here aims to identify the different types of contaminants in raw/pasteurized cow's milk and analyze the application of alternative processes for the elimination or degradation of contaminants.\n\n\n2. Contaminants present in cow's milk\n\nThere are several hazards of contamination of cow's milk, ranging from biological to chemical compounds. The risk of biological contamination of cow's milk derives mainly from cattle milking due to the exposure of udders to the environment, equipment, storage, dirty pipes, and others.10 Chemical contamination of cow's milk comes from several sources: application of agrochemicals,11 use of legal or illegal veterinary products,12 feed and forages contaminated with natural toxins,13 or through the improper use of chemicals during milk production, processing and packaging stages.14\n\nFigure 1 shows the direct and indirect pathways for contaminants entry into bovine milk.\n\nIndirect contamination is associated with the ingestion of contaminants both from the environment and from substances of veterinary use. The most common environmental contaminants are mycotoxins, pesticides, and metals consumed by cattle through feed, forages, and water. In addition, antibiotics and hormones are administered to the cow orally, by injection, or as intramammary infusions to treat diseases, promote animal growth and increase milk production.5 On the other hand, direct contamination occurs during milk processing from milking, handling, storage and even pasteurization. During the industrialization process, milk comes into contact with metals, residues of cleaning products, mycotoxins, among others.\n\nFor better analysis and understanding, the classification of contaminants according to the origin is microbial contaminants and chemical contaminants (Figure 2).\n\nAbout 14.57% of the literature reports contamination of cow's milk by pathogenic microorganisms. Although the objective of the pasteurization process is the elimination of these microorganisms, there is evidence of their presence in pasteurized milk, which will be presented later. Although pathogenic microorganisms are considered the main hazard that threatens the safety of milk, they do not represent the highest percentage of reported cases. The contaminants that have been more reported in the literature are of chemical origin (Figure 2). Among chemical contaminants, metals, pesticides, and antibiotics stand out. Among chemical contaminants, the most reported are heavy metals (22.18%), pesticides (22.05%), and antibiotics (22.18%); due to bad practices in agriculture and cattle. Although reports of mycotoxins in milk are relatively low (9.97%), they are of great importance due to the increase in reported cases of contamination with Aflatoxin M1 (AFM1). The International Agency for Research on Cancer has classified AFM1 as a carcinogenic substance.15 This means that the food safety of milk is at risk, as any of these compounds compromise the health of the final consumer. Below is a detailed classification of the different types of contaminants present in both raw and pasteurized milk and the negative effects they have on consumer health.\n\nThe presence of several pathogenic microorganisms has been reported in raw and pasteurized cow's milk (Table 1). Microbial contamination of raw milk can be due to diseases such as mastitis, improper handling on production farms, milking equipment, water sources, and feeding of cattle, utensils, and equipment used for milk storage on the farm or during transport.16 Likewise, poor hygienic practices within the dairy industry can lead to the formation of biofilms on the sprinklers of cooling systems, pipes, cooling tanks, storage, and transport tanks. The contact of pasteurized milk with these surfaces increases the risk of contamination with pathogenic microorganisms, posing a danger to the consumer and the quality of the product.17\n\nAccording to Table 1, Most cases of contamination are recorded in raw milk due to inadequate milking, processing, storage, and transport conditions. On the other hand, although few studies report the presence of microorganisms in pasteurized milk, it is doubtful that it is an efficient process for their elimination. The main types of microorganisms present in milk are bacteria, yeasts, and molds, which represent the different types of microorganisms present in cow's milk. The presence of Corynebacteria, Staphylococcus, Streptococcus, Bacillus, and Micrococcus species has been evidenced in the teat of dairy cattle.38,39 These microorganisms have also been identified in cow's milk,27,28,40,41 demonstrating that during milking, milk can become contaminated by contact with the cow's teat under unhygienic conditions. On the other hand, as a result of mastitis, Staphylococcus and Streptococcus species have been identified in bovine milk samples,42,43 with Staphylococcus aureus being the main cause of mastitis.43 The presence of Enterobacteriaceae, Pseudomonas spp., Staphylococcus spp., and lactic acid bacteria has been identified in the equipment used for milking.17 It is evident that the conditions under which milk is obtained on farms are not the most adequate because these different microorganisms are found in cow's milk.33,42,44,45\n\nConsumption of milk contaminated by pathogenic microorganisms such as Campylobacter, Salmonella, Yersinia, E. coli, Listeria, and S. aureus can cause muscle and stomach pain, gastrointestinal diseases with diarrhea, fever, and nausea.31 These microorganisms are commonly found in the intestinal flora or in the udder of cows, thus facilitating milk contamination.31 In addition, Campylobacter spp. and E. Coli O157:H7 are capable of producing Guillain-Barrés syndrome and hemolytic uremic syndrome, respectively.46\n\nFor a more detailed analysis, the chemical contaminants found in cow's milk have been classified into five groups: pesticides, metals, antibiotics, mycotoxins, and hormones (Table 2).\n\n2.2.1 Pesticides\n\nA variety of pesticide residues in detectable amounts in raw milk, pasteurized, and UHT (ultra-high temperature) milk has been reported by several authors. This is due, among other factors, to the lipophilic properties and resistance to biodegradation of these types of contaminants.8 There are three possible forms in which pesticides can enter the animal's body172: (i) through contaminated water, (ii) through the pores of the skin when the animal is sprayed or soaked to treat ectoparasites, and (iii) through contaminated feed and forage, the latter being the main source of entry.\n\n(i) The presence of organophosphorus pesticide residues (malathion, methyl-parathion, diazinon, ethion) was identified. The average concentrations detected were 0.032-0.78, 0.13, 0.32-0.74, 0.010 μg/L for malathion, methyl-parathion, diazinon and ethion, respectively.62,173 Fipronil and chlorpyrifos were other pesticides found in water samples supplied to livestock.174,175 Ashoub & Azam176 identified DDT (Dichloro diphenyl trichloroethane), aldrin, heptachlor epoxide, lindane, methoxychlor, diazinon, and deltamethrin in water samples from cattle farms. These same compounds have been identified in cattle drinking water and in cow's milk.11,47,51,54,55,62,65,177–180 This verifies that water contaminated by pesticides and supplied to cattle is one of the main routes of contamination of raw cow's milk.\n\n(ii) According to the analysis of Table 2, Claborn et al.181 report the presence of malathion residues in cow's milk after cattle were sprayed with this pesticide for the treatment of ectoparasites. Malathion was found to be completely secreted from the udder 24 hours after application. In contrast to malathion, lindane was reported not to be completely excreted in milk until seven days after application to the cow's skin.182 Residues of chlorpyrifos and ethion have been found in cow milk up to 24 and 72 hours after application, respectively.183 This confirms that skin contaminated with these pesticides is another route of contamination of raw cow's milk.\n\n(iii) In forage, concentrations of 0.02 mg kg-1 of DDT residue were reported.184 The presence of cypermethrin, chlorpyrifos, cyhalothrin, and deltamethrin in forage was reported in a range of mean concentrations between 1.03-6.01 ng g-1. In addition to the presence of pesticides in forages, residues of lindane, DDT, fenvalerate, ethion, malathion, profenofos were also reported in feed. The mean concentrations of these varied in the range of 0.63-4.05 ng g-1.175 The presence of deltamethrin in feed was also reported in a concentration range of 41.99-381.30 μg kg-1.185 Another investigation revealed the presence of malathion, dimethoate, methyl-parathion, diazinon in the feed fed to cattle. The range of detected concentrations was between 0.01-80.45 μg L-1.62 All the contaminants reported in forage and feed were also detected in cow's milk.11,54,55,57,60–62,177,179,186 Thus, like water, pesticide-contaminated forage and feed are a route of contamination as they are directly ingested by cattle and excreted through cow's milk.\n\nPesticides are one of the most commonly found contaminants, not only in raw cow's milk but also after the pasteurization and UHT process. Their presence in milk, even below the maximum permitted levels, represents a health risk to the consumer. It is related to Hodgkin's disease (HD), non-Hodgkin's lymphoma (NHL), Parkinson's disease, endocrine disruption, respiratory and reproductive disorders, among others.187\n\nIt is important to note that organochlorine pesticides such as hexachlorocyclohexane, dichloro diphenyl trichloroethane, and endosulfane are still present despite having been banned since the 1970s because of their high persistence in the environment and their harmful effects on human health,188 are still detected in cow's milk. This indicates that they are still used in agriculture and animal husbandry. With a few exceptions (cyhalothrin, cypermethrin, fenvalerate, deltamethrin, permethrin, and diazinonella), the vast majority of pesticides found in cow's milk are not regulated by Codex and the EU. This demonstrates the low efficiency of the regulatory controls of these contaminants in the unprocessed and post-processed product, leading to an inefficient safety of this food product.\n\n2.2.2 Metals\n\nAlthough metals are found in the environment either naturally or due to industrial and/or agricultural activities, there are several routes by which they reach the milk. Namely, ingestion of contaminated food, fodder, and/or contaminated drinking water. In the soil, they are absorbed by many crop plant species, which, when ingested by animals, are transferred to the lactating glands and finally excreted in milk.172 Equipment used in the dairy industry is another source of contamination directly to milk with metals such as chromium and nickel.189 Heavy metals such as cadmium, lead, mercury, and arsenic reach milk by indirect contact through feed consumed by cattle.189 Although the literature does not report the presence of metals in water or fodder destined for cattle, as well as in pesticides, these can be another of the main routes of contamination.\n\nSeveral heavy metals have been reported in the literature to be found in raw cow's milk. The metals least found in studies of raw cow milk are tin and molybdenum. These elements are not abundant in nature, and their presence in fodder or water for animal consumption will depend on soil characteristics, while the most reported are lead, cadmium, copper, and zinc, due to environmental pollution produced by man mainly in industrial activities.79,190 Minerals such as Fe, Cu, and Zn are necessary for various biological functions. However, high concentrations of these minerals have negative effects on human health.96 Lead is one of the non-essential metals classified as carcinogenic to humans by the International Agency for Research on Cancer.191 Cadmium is associated with the formation of human lung, kidney, breast, prostate, urinary tract cancer because it affects cell proliferation, differentiation, and other cellular activities.192\n\nNone of the heavy metals reported in the literature consulted have established maximum residue limits (MRLs) by Codex193 and the EU.194 However, these contaminants are known to represent a high risk to human health. Stricter control measures should be adopted in the dairy industry, considering that cow's milk is one of the most consumed products by humans worldwide.\n\n2.2.3 Antibiotics\n\nAntibiotics are used in livestock activities in three basic ways: therapeutic, prophylactic, and growth promoters. About 80% of dairy cattle are subjected to antibiotic treatments on at least one occasion throughout their lives, mostly used as growth promoters and for the treatment of various diseases such as mastitis, arthritis, respiratory diseases, gastrointestinal diseases, and bacterial infections.195 Cows eliminate antibiotics and their metabolites through milk, depending on the dose and route of application, level of milk production, type and degree of mammary disease, and time between treatment and milking. On the other hand, oral, intramuscular, or intravenous administration is less important from the point of view of milk hygiene than intramammary application. However, intramammary antibiotics are easy to apply and generally cheaper, so they are preferred in dairy farms.\n\nThe most common disease in dairy cows is mastitis, whose treatment includes the wide use of tetracyclines, β-lactams, oxytetracycline, difloxacin, among others, being the β-lactams of greater application.8 Within the latter group, the most employed are penicillin, ampicillin, and amoxicillin.196 According to the literature, the presence of antibiotics in milk has been evidenced, highlighting tetracycline, oxytetracycline, penicillin, and amoxicillin.103,124,197,198 While other antibiotics less reported in milk were rifamixin, gatifloxacin, spiramycin, and lomeflaxacin, with no indication in the studies of the purpose of their application in cattle.101,112,126,127\n\nThe consumption of contaminated milk with antibiotic residues is an emerging public health problem worldwide. Therefore, it is important to control the presence of antibiotic residues in food to avoid the appearance of resistance to these antibiotics in humans. The presence of antibiotics at concentrations even below the MRL in milk can cause undesirable effects on human health such as ototoxicity and nephrotoxicity,199 endocrine disruption,200 hypersensitivity, and especially bacterial resistance.130 According to the literature consulted, 43 antibiotics present in cow's milk have been identified, of which 18 are not regulated by Codex193 and EU standards.194\n\nConsidering that the use of antibiotics in cattle generates residues in milk, their excessive use should be avoided, and the elimination times before milking should be respected in order to avoid the presence of these contaminants.\n\n2.2.4 Mycotoxins\n\nThe quality of food products is commonly affected by toxin contamination, of which 60 to 80 % are caused by mycotoxins.201 This means a risk for human health and great economic losses in the industrial sector.\n\nMycotoxins are natural contaminants produced by Aspergillus, Penicillium, and Fusarium fungi,154 the most prominent being AFM1, which results from the metabolism of aflatoxin B1 in the liver of contaminated animals.15,143 In the 1960s, the first reported case of aflatoxin contamination was reported for the first time, beginning the concern for this type of contaminant. Even during this decade, high consumption of feed contaminated by this mycotoxin was reported, which led to indirect contamination of cow's milk for consumption, compromising the safety of this product.202 Therefore, it is considered that the main routes of entry of mycotoxins into milk are contaminated crops and feed ingested by cows.136\n\nIt is known that approximately 0.3-6.2% of AFB1 (Aflatoxin B1) present in animal feed is converted to AFM1.15 This mycotoxin is neither degraded nor removed by industrial food processes such as pasteurization and sterilization, nor by the cooking of feed.203 This represents a difficult problem to deal with at the industrial level due to the stability of mycotoxins in general to thermal, physical, and chemical treatments.204\n\nAFM1 mycotoxin is the only regulated by Codex193 and EU194 and the most reported in cow's milk according to the literature. However, other abundant mycotoxins have been identified in this food product, such as ochratoxin A and zearalenone. The fungi of the genus Aspergillus and Penicillium produce Ochratoxin A, while fungi of the genus Fusarium produces zearalenone, commonly found in cattle feed.138 On the other hand, aflatoxin G2, aflatoxin G1, aflatoxin B2, and zearalanol show a lower incidence in cow's milk. The literature on the effects on human health associated with the ingestion of mycotoxin-contaminated milk is scarce or almost non-existent, unlike AFM1. Therefore, studies on this type of contaminants should be expanded.\n\n2.2.5 Hormones\n\nThe use of hormones in the livestock industry increases production yields and medical treatments. Their fat-soluble characteristics favor their high persistence and presence in cow's milk due to the high-fat contents.156 Therefore, the supply of hormones to cattle represents a form of direct contamination that, like other contaminants, is excreted through milk. However, the European Union banned the use of hormones through the Directive 96/22/EC, and enforcement is regulated by Directive 96/23/EC.165\n\nPrednisolone in combination with amoxicillin and clavulanic acid is used to treat mastitis in cows' udders,205 being an access route of this contaminant to milk. The 17β-estradiol and progesterone, with the highest presence in cow milk, are sex hormones widely used to induce lactation, improve fertility and synchronize the estrous cycle.8,168 The hormones least found in studies in milk were testosterone, somatostatin, and cortisone. The presence of estrogens in cow's milk has been linked to diseases such as breast cancer206 and conditions in the gastrointestinal tract.156 Other diseases associated with the presence of hormones in cow's milk have included acne, prostate cancer, uterine cancer, and male reproductive disorders.167\n\nTable 2 shows that several hormones are frequently present in cow's milk, with prednisolone being the only one regulated by the EU.194 This indicates that regulations should be established for different hormones considering that they are the chemical compounds mostly used to increase milk production yield to preserve quality and consumer safety.\n\n\n3. Pasteurization process in cow's milk\n\nThe principles and name of pasteurization come from the studies of the French scientist Louis Pasteur. His interest in milk and other food products was due to their putrefaction, which he later attributed to the growth of undesirable microorganisms.207 Several pathogenic microorganisms are found in raw milk: Pseudomonas, Enterobacter, Bacillus, Clostridium, Microbacterium, and Micrococcus. Pathogenic microorganisms in cow's milk have been linked to infectious diseases such as campylobacteriosis, salmonellosis, yersiniosis, listeriosis, tuberculosis, brucellosis, staphylococcal enterotoxin intoxication, streptococcal infections, and Escherichia coli O157: H7 infection.208\n\nIt was not until the end of the 1880s that heat treatment began to be used to commercialize milk. This arose with the main objective of inactivating Mycobacterium tuberculosis, the cause of tuberculosis in humans associated with the consumption of raw milk. Thus, pasteurization became a process universally employed by developed countries after World War II. However, there is evidence that not all pathogenic microorganisms can be eliminated during pasteurization, such as Staphylococcus aureus, micrococci, Streptococcus spp, and Bacillus.209 Which calls into question the efficiency of this process.\n\nThe US Food and Drug Administration (FDA) establishes a maximum limit for bacteria in raw cow's milk of 100,000 cfu ml-1 and 20,000 cfu ml-1 for pasteurized milk.209\n\nPasteurization is a technology classified on the basis of operating temperatures and exposure times as follows: LTLT, HTST, and UHT. Low-temperature long-time pasteurization (LTLT) uses a minimum temperature of 62.8°C and a minimum time of 30 min. High-temperature short-time pasteurization (HTST) uses a minimum temperature of 71.1°C, a minimum time of 15 seconds, and ultra-high temperature pasteurization (UHT) works at a minimum of 135°C and during a minimum time of 1 second.210 Pasteurized milk under UHT conditions can be stored for several months without refrigeration.211 Whereas the shelf life of pasteurized milk ranges from 10 to 20 days when kept under refrigerated conditions below 6.1°C.212\n\nIt has been shown that the application of pasteurization denatures proteins with bacteriostatic capacity, as is the case of lactoferrin. This is a glycoprotein that binds iron, and its complete denaturation has been evidenced losing its inhibitory capacity on Escherichia coli under UHT conditions.213 For this reason, it is suggested that heat treatment should be applied below 75°C to avoid denaturation of proteins with bacteriostatic capacity and at the same time cause inactivation of pathogenic microorganisms.213\n\nOn the other hand, the HTST process degrades up to 20% of the vitamins (B1, B6, B12, and C) present in milk.214 This evidence shows that, although pasteurization and UHT have been widely used to eliminate pathogenic microorganisms, it is not entirely efficient for this purpose. There are even losses of milk mineralization, varying its nutritional composition.\n\nThe presence of microbial contaminants in different samples of pasteurized milk shows that, although pasteurization aims to eliminate microorganisms present in milk, it is not totally effective. Moreover, with the appearance of other contaminants, the quality of milk no longer depends only on the presence of microorganisms. It is, therefore, necessary to study other methods of decontamination to ensure the safety and health of consumers.\n\n\n4. Alternative methods for the treatment of cow's milk\n\nInternational regulations require maximum limits for microbial and chemical contaminants to ensure the quality of drinking milk. Pasteurization is a technology widely used in the dairy industry. However, it is exclusive for the elimination of microbial contaminants. The literature mentions alternatives for eliminating specific microbial and chemical contaminants (Table 3).\n\nSupercritical carbon dioxide has been used as an inactivating agent for E. coli, where the greatest reduction in the content of microorganisms was observed during a residence time of 20 minutes, achieving almost complete inactivation after 70 minutes.215 Complete inactivation of coliforms, molds, and yeasts was achieved, while a maximum reduction of aerobic bacteria of 4.96 log was obtained using high-pressure carbon dioxide.221 Using a thin-film UV-C (Ultraviolet-C) reactor with flow-guiding elements allowed a 4.58 log and 3.19 log reduction for E. coli and L. innocua, respectively.216 Makarapong et al.218 employed a UV-C reactor for the inactivation of aerobic bacteria achieving a 4.60 log and 4.70 log reduction at 48W and 39W, respectively. UV-C lamp wattage did not significantly influence the fat concentration in the milk, which means that it is necessary to improve the method to guarantee an effective reduction of these microorganisms if milk transport time exceeds two hours without cooling. It was verified that L. monocytogenes was completely inactivated in milk with ozone for 15 minutes. However, nutritional values were affected.219 Exposure of milk to Nd:YAG laser did not alter the physicochemical properties of milk, but the percentage of reduction was low for E.coli (30%), Salmonella sp (25%), yeasts (47%), and Lactobacillus sp (30%).223 The combination of ultrasound with hydrogen peroxide and an active lactoperoxidase system was able to guarantee the microbial quality of milk as it was able to completely inactivate Staphylococcus aureus, Listeria monocytogenes, Lactobacillus plantarum, Lactobacillus pentosus, Salmonella Typhimurium, Escherichia coli, and Pseudomonas fluorescens at 10 minutes at an amplitude of 125 μm.220 The application of ultrasound in combination with variations in temperature, time, and constant pressure (manothermosonication) achieved minimal reductions of up to 1.6 log CFU/ml for E. coli and P.fluorescens and 1.05 log CFU/ml for S. aureus. Further studies are needed to ensure effective inactivation using manothermosonication.225 The application of high pressures (400-600MPa) effectively inactivated (5 log CFU/ml) E. coli, Salmonella and L. monocytogenes, Enterobacteriaceae, lactic acid bacteria, and Pseudomonas spp.222 One of the most widely used methods for the inactivation of microorganisms in cow's milk is pulsed electric fields (PEF). This method was applied for the inactivation of E.coli and L. innocua, achieving a reduction of 2 log CFU/ml.217 It was found that combining this method with preheating at 50°C achieved a 5-6 log CFU/ml reduction of Pseudomonas aeruginosa and a total reduction of E. coli, S. aureus, and L. innocua.224\n\nBiosorption methods employing the use of microorganisms prove to be efficient in the removal of pesticides, metals, and mycotoxins. Biosorption with lactic acid bacteria managed to eliminate organophosphate pesticides from cow's milk, being more effective for chlorpyrifos, fenitrothion, and malathion, whose degradation constants were greater than 0.018 h-1. On the other hand, diazinon and methyl parathion were more resistant when applying of the different strains of lactic acid bacteria separately and in combination. The degradation rate constants were correlated with the measurement of phosphatase activity, and it was found that the lower the phosphatase activity, the lower the degradation constant.226 The same method was applied for this group of contaminants finding that dimethoate and methyl parathion were the most stable with the lowest degradation rate constants (0.0165-0.0184 and 0.0213 h-1, being more efficient for the removal of malathion with higher degradation rate constants (0.0218-0.0420 h-1).228 Although the application of lactic acid bacteria was shown to be an effective method for removing diazinon, dimethoate, and methyl parathion in cow's milk it was not very selective since it cannot eliminate all the organophosphates studied.\n\nBiosorption with Saccharomyces Cerevisiae allowed the removal of 70% of lead, mercury, and cadmium metals.230,233–235 The removal percentage was higher when Lactobacillus Acidophilus was used, eliminating 80, 75, and 72%, respectively.231,232 The use of Saccharomyces cerevisiae and Lactobacillus helveticus removed AFM1 from milk by an as yet unknown binding mechanism.243 A combination of probiotic bacteria with yeast species managed to remove 90.88% of AFM1 within 72 hours.245 This percentage of removal was higher than that obtained in another study (19-61%).246 By applying a biofilm of Lactobacillus rhamnosus, an AFM1 removal of 60.74% was achieved. Despite that, the method is not a viable alternative for application because a reduction in the percentage of fat and total dry matter was observed.247\n\nBiosorption methods employing microorganisms (Lactobacillus acidophilus and Saccharomyces cerevisiae) are efficient for removing heavy metals in cow's milk (lead, mercury, copper, and cadmium). However, they require a minimum fermentation period of 4 days. When using lactic acid bacteria to degrade organophosphorus pesticides, a minimum fermentation period of 24 hours is required. These times would represent economic losses for the industry, and given the existing world demand for milk, it would be almost impossible to apply them on a large scale.\n\nAdsorption methods prove to be efficient for removing metals, antibiotics, and mycotoxins. By adsorption with diacrylate Pluronic P123 (P123-DA) hydrogels removed about 85.3% and 81.9% of Pb2+, and Hg2+ ions, respectively.229 Resins have been another adsorbent used in the adsorption of heavy metals in cow's milk. IMAC HP resin was described for the removal of copper ions (76.89%).92 Tetracycline, oxytetracycline, chlortetracycline, and doxycycline have been removed by adsorption on a molecularly imprinted polymer, achieving 81.83, 95.47, 96.44, and 93.25% removal, respectively.239 A photocatalytic-fluorescent polymer, produced from graphene oxide and bismuth phosphate with molecular magnetic imprinting, allowed ciprofloxacin's complete degradation.240 Bodbodak et al.,242 developed a molecularly imprinted polymer coated on the surface of a stainless-steel plate as an adsorbent material for the decontamination of AFM1 in cow's milk. This method was able to remove 87.3 to 96.2% of AFM1 without causing a change in the physicochemical properties of the milk. Adsorption with kaolin and natural calcium bentonite clay for adsorption was able to remove AFM1 by 86.1-93.3% and 93.7-97.7%, respectively. It was observed that no change in the nutritional properties of milk would occur.244 Despite this, few studies have been reported in cow's milk. Therefore, there are not enough to consider its application at the industrial level.\n\nOther methods less reported in the literature were also applied for the removal of pesticides and antibiotics. The ultrasonic treatment proved to be effective for the degradation of 97.10% of methyl parathion. However, this method is limited by the generation of degradation products with toxic effects.227 For the elimination of antibiotics in cow's milk, methods such as ozonation have been applied, with about 95% degradation for amoxicillin, doxycycline, ciprofloxacin, and sulfadiazine.236 Electrochemical oxidation applied for the removal of small concentrations of chlortetracycline, cefazolin,237 and oxytetracycline238 was also described. Gamma radiation was also found to be effective for the removal of amoxicillin, ciprofloxacin, and doxycycline by 90% in cow's milk samples.241 However, of all the antibiotics detected in cow's milk, they have only been tested for the elimination of amoxicillin, doxycycline, ciprofloxacin, sulfadiazine, chlortetracycline, cefazolin, y tetracycline. More studies are needed to validate the application of these methods for the decontamination of cow's milk.\n\nIt has not been demonstrated that a single method is capable of eliminating different groups of contaminants, as is the case of pasteurization for microbial contaminants. Despite the wide use of hormones in the cattle industry and their consequent generation of traces in cow's milk, no removal methods have been reported for them. The alternative methods studied to date have been applied on an industrial scale, and many of them alter the nutritional properties of milk. The fact that most of these chemical contaminants are not regulated by standards does not oblige the dairy industry to use alternative methods to pasteurization. Nor is it economically viable to use a different method for the elimination of each contaminant present in milk. However, to guarantee the safety of milk, it is essential to study processes that complement pasteurization and can eliminate pathogenic microorganisms and chemical contaminants.\n\n\n5. Conclusions and future prospects\n\nThe presence of contaminants in raw cow's milk (many of them banned) is an indication that they are currently used illegally in both agriculture and animal husbandry. Although the presence of contaminant residues in milk represents a health risk to the consumer, there are no MRLs established for all of them. In addition, pasteurization processes are not efficient for the degradation or elimination of the different contaminants addressed.\n\nAlthough, the literature exposes alternative methods for removing various contaminants in milk, they are still not sufficient nor applied on an industrial scale. Instead, they have been applied individually or in very small families of contaminants. There are no evidence or results concerning the interactions between them or with intermediate products formed on cow's milk, nor changes in the organoleptic properties. A particular case is hormones, which although they are a direct source of contamination, with evidence of their presence in raw, pasteurized, and UHT milk, the literature does not report specific elimination methods for these types of contaminants.\n\nHowever, alternative methods have proven to be efficient in degrading several contaminants present in milk. Based on this hypothesis, it is suggested to deepen the application of these methods, including the study of interactions between different families of contaminants, application of new materials, or modification of existing ones. Studies on toxicity or changes in organoleptic properties. In this sense, the field of nano-biotechnology, nano-fibers, nano-membranes, biochar, MOF's (metal-organic framework), among others, could play a relevant role, guaranteeing the safety of the milk consumed, and consequently, a better quality of life for consumers.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgments\n\nThe authors would like to thank the Universidad Técnica de Manabí for the support offered.\n\n\nReferences\n\nO’Mahony JA, Fox PF: Milk: An Overview. Milk Proteins. Singh H, Boland M, Thompson A, editors. San Diego: Academic Press; 2014; p. 19–73. Publisher Full Text\n\nFAO: Food Outlook. Trade and Market Division of FAO. Food and Agriculture Organization of the United Nations; 2010 [cited 2020 Sep, 29]. Reference Source\n\nFAO: Food Outlook-Biannual Report on Global Food Markets. 2019 [cited 2020 Sep, 27]. Reference Source\n\nFox PF: Milk\|Bovine Milk. Encyclopedia of Dairy Sciences. Fuquay JW, editor. 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PubMed Abstract \| Publisher Full Text\n\nAzzouz A, et al.: Simultaneous determination of 20 pharmacologically active substances in cow's milk, goat's milk, and human breast milk by gas chromatography–mass spectrometry. J. Agric. Food Chem. 2011; 59(9): 5125–5132. PubMed Abstract \| Publisher Full Text\n\nSocas-Rodríguez B, et al.: Multiclass analytical method for the determination of natural/synthetic steroid hormones, phytoestrogens, and mycoestrogens in milk and yogurt. Anal. Bioanal. Chem. 2017; 409(18): 4467–4477. PubMed Abstract \| Publisher Full Text\n\nKaklamanos G, Theodoridis G: Rapid multi-method for the determination of growth promoters in bovine milk by liquid chromatography-tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2013; 930: 22–29. PubMed Abstract \| Publisher Full Text\n\nFlorez DHÂ, de Oliveira HL , Borges KB: Polythiophene as highly efficient sorbent for microextraction in packed sorbent for determination of steroids from bovine milk samples. Microchem. J. 2020; 153: 104521. Publisher Full Text\n\nRegal P, Cepeda A, Fente C: Development of an LC-MS/MS method to quantify sex hormones in bovine milk and influence of pregnancy in their levels. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2012; 29(5): 770–779. PubMed Abstract \| Publisher Full Text\n\nTrapiella-Alfonso L, et al.: Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk. Biosens. Bioelectron. 2011; 26(12): 4753–4759. PubMed Abstract \| Publisher Full Text\n\nTan X-t, et al.: Analysis of 13 kinds of steroid hormones in raw milk using modified QuEChERS method combined with UPLC-QTOF-MS. J. Integr. Agric. 2016; 15(9): 2163–2174. 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Publisher Full Text\n\nLosada A, et al.: Organochlorine pesticide residues in bovine milk from León (Spain). Sci. Total Environ. 1996; 181(2): 133–135. Publisher Full Text\n\nJohn PJ, Bakore N, Bhatnagar P: Assessment of organochlorine pesticide residue levels in dairy milk and buffalo milk from Jaipur City, Rajasthan, India. Environ. Int. 2001; 26(4): 231–236. PubMed Abstract \| Publisher Full Text\n\nClaborn HV, et al.: Pesticide Residues, Malathion in Milk and Fat from Sprayed Cattle. J. Agric. Food Chem. 1956; 4(11): 941–942. Publisher Full Text\n\nBushland RC, et al.: Contamination of meat and milk by chlorinated hydrocarbon insecticides used for livestock pest control. J. Econ. Entomol. 1950; 43(5): 649–652. Publisher Full Text\n\nRodrigues FM, et al.: Development of a headspace solid-phase microextraction/gas chromatography–mass spectrometry method for determination of organophosphorus pesticide residues in cow milk. Microchem. J. 2011; 98(1): 56–61. 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Publisher Full Text\n\nFischer WJ, et al.: CONTAMINANTS OF MILK AND DAIRY PRODUCTS\|Environmental Contaminants. Encyclopedia of Dairy Sciences. Roginski H, editor. Oxford: Elsevier; 2002; p. 525–533. Publisher Full Text\n\nLicata P, et al.: Levels of “toxic” and “essential” metals in samples of bovine milk from various dairy farms in Calabria, Italy. Environ. Int. 2004; 30(1): 1–6. PubMed Abstract \| Publisher Full Text\n\nIARC W: World Cancer Report 2008. World Health Organization; 2008; vol. 29. .\n\nZhou X, et al.: Relationships between Pb, As, Cr, and Cd in individual cows' milk and milk composition and heavy metal contents in water, silage, and soil. Environ. Pollut. 2019; 255(Pt 2): 113322. PubMed Abstract \| Publisher Full Text\n\nFAO: Maximum residue limits (MRLs) and risk management recommendations (RMRs) for residues of veterinary drugs in foods. CX/MRL 2–2018. London: FAO; 2018.\n\nEuropea, C: Reglamentos UE N 37/2010 de la Comisión de 22 de diciembre de 2009. Diario Oficial de La Unión Europea; 2010.\n\nBacanlı M, Başaran N: Importance of antibiotic residues in animal food. Food Chem. Toxicol. 2019; 125: 462–466. PubMed Abstract \| Publisher Full Text\n\nDe Oliveira AP, et al.: Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and the United States. J. Dairy Sci. 2000; 83(4): 855–862. PubMed Abstract \| Publisher Full Text\n\nGhidini S, et al.: Residues of beta-lactam antibiotics in bovine milk: confirmatory analysis by liquid chromatography tandem mass spectrometry after microbial assay screening. Food Addit. Contam. 2003; 20(6): 528–534. PubMed Abstract \| Publisher Full Text\n\nNavratilova P, et al.: Occurrence of tetracycline, chlortetracycline, and oxytetracycline residues in raw cow’s milk. Czech J. Food Sci. 2009; 27(5): 379–385. Publisher Full Text\n\nShi Q, et al.: Utilization of a lateral flow colloidal gold immunoassay strip based on surface-enhanced Raman spectroscopy for ultrasensitive detection of antibiotics in milk. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2018; 197: 107–113. PubMed Abstract \| Publisher Full Text\n\nMarrugo-Padilla A, Méndez-Cuadro D, Rodríguez-Cavallo E: Combined tetracycline and pyrethroid residues increases protein carbonylation in bovine milk. Int. Dairy J. 2020; 107: 104708. Publisher Full Text\n\nEskola M, et al.: Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited ‘FAO estimate’of 25%. Crit. Rev. Food Sci. Nutr. 2020; 60(16): 2773–2789. PubMed Abstract \| Publisher Full Text\n\nBecker-Algeri TA, et al.: Mycotoxins in Bovine Milk and Dairy Products: A Review. J. Food Sci. 2016; 81(3): R544–R552. PubMed Abstract \| Publisher Full Text\n\nSweeney MJ, Dobson AD: Mycotoxin production by Aspergillus, Fusarium and Penicillium species. Int. J. Food Microbiol. 1998; 43(3): 141–158. Publisher Full Text\n\nMarin S, et al.: Mycotoxins: occurrence, toxicology, and exposure assessment. Food Chem. Toxicol. 2013; 60: 218–237. Publisher Full Text\n\nLiu Y, et al.: Simultaneous detection and comparative pharmacokinetics of amoxicillin, clavulanic acid and prednisolone in cows' milk by UPLC-MS/MS. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2016; 1008: 74–80. PubMed Abstract \| Publisher Full Text\n\nGanmaa D, Sato A: The possible role of female sex hormones in milk from pregnant cows in the development of breast, ovarian and corpus uteri cancers. Med. Hypotheses. 2005; 65(6): 1028–1037. PubMed Abstract \| Publisher Full Text\n\nWilbey RA: HEAT TREATMENT OF FOODS\|Principles of Pasteurization. Encyclopedia of Food Microbiology. Batt CA, Tortorello ML, editors. Oxford: Academic Press; 2014; p. 169–174. Publisher Full Text\n\nSteele JH: History, trends, and extent of pasteurization. J. Am. Vet. Med. Assoc. 2000; 217(2): 175–178. PubMed Abstract \| Publisher Full Text\n\nÖzer B, Yaman H: MILK AND MILK PRODUCTS\|Microbiology of Liquid Milk. Encyclopedia of Food Microbiology. Batt CA, Tortorello ML, editors. Oxford: Academic Press; 2014; p. 721–727. Publisher Full Text\n\nRyser ET: Liquid Milk Products\|Pasteurization of Liquid Milk Products: Principles, Public Health Aspects. Encyclopedia of Dairy Sciences. Fuquay JW, editor. San Diego: Academic Press; 2011; p. 310–315. Publisher Full Text\n\nLorenzen PC, et al.: A survey of the quality of extended shelf life (ESL) milk in relation to HTST and UHT milk. Int. J. Dairy Technol. 2011; 64(2): 166–178. Publisher Full Text\n\nSarkar S: Microbiological considerations: pasteurized milk. Int. J. Dairy Sci. 2015; 10(5): 206–218. Publisher Full Text\n\nXiong L, et al.: Effect of heat treatment on bacteriostatic activity and protein profile of bovine whey proteins. Food Res. Int. 2020; 127: 108688. PubMed Abstract \| Publisher Full Text\n\nMeunier-Goddik L, Sandra S: Liquid Milk Products: Pasteurized Milk. Reference Module in Food Science. Elsevier; 2016. Publisher Full Text\n\nCeni G, et al.: Continuous inactivation of alkaline phosphatase and Escherichia coli in milk using compressed carbon dioxide as inactivating agent. J. CO2 Util. 2016; 13: 24–28. Publisher Full Text\n\nBarut Gok S, et al.: Inactivation of E. coli and L. innocua in milk by a thin film UV-C reactor modified with flow guiding elements (FGE). Int. J. Food Microbiol. 2021; 343: 109105. PubMed Abstract \| Publisher Full Text\n\nSharma P, et al.: Reduction of bacterial counts and inactivation of enzymes in bovine whole milk using pulsed electric fields. Int. Dairy J. 2014; 39(1): 146–156. Publisher Full Text\n\nMakarapong D, et al.: Development of an innovative apparatus using UV-C for controlling the number of microorganisms in raw milk after milking. Int. J. Dairy Technol. 2020; 73(1): 301–305. Publisher Full Text\n\nSheelamary M, Muthukumar M: Effectiveness of ozone in inactivating Listeria monocytogenes from milk samples. World Journal of Young Researchers. 2011; 1(3): 40–44.\n\nShamila-Syuhada AK, et al.: Inactivation of microbiota and selected spoilage and pathogenic bacteria in milk by combinations of ultrasound, hydrogen peroxide, and active lactoperoxidase system. Int. Dairy J. 2016; 61: 120–125. Publisher Full Text\n\nHongmei L, et al.: Inactivation of microorganisms naturally present in raw bovine milk by high-pressure carbon dioxide. Int. J. Food Sci. Technol. 2014; 49(3): 696–702. Publisher Full Text\n\nStratakos AC, et al.: Effect of high pressure processing on the safety, shelf life and quality of raw milk. Innovative Food Sci. Emerg. Technol. 2019; 52: 325–333. Publisher Full Text\n\nYasmin N, et al.: Inactivation of foodborne pathogens on food packaging and in cow milk by exposure to a Nd:YAG laser. Can. J. Phys. 2017; 95(7): 662–669. Publisher Full Text\n\nSharma P, et al.: Bacterial inactivation in whole milk using pulsed electric field processing. Int. Dairy J. 2014; 35(1): 49–56. Publisher Full Text\n\nCregenzán-Alberti O, et al.: Suitability of ccRSM as a tool to predict inactivation and its kinetics for Escherichia coli, Staphylococcus aureus and Pseudomonas fluorescens in homogenized milk treated by manothermosonication (MTS). Food Control. 2014; 39: 41–48. Publisher Full Text\n\nZhang YH, et al.: Enhanced degradation of five organophosphorus pesticides in skimmed milk by lactic acid bacteria and its potential relationship with phosphatase production. Food Chem. 2014; 164: 173–178. PubMed Abstract \| Publisher Full Text\n\nYuan S, et al.: Degradation of parathion methyl in bovine milk by high-intensity ultrasound: Degradation kinetics, products and their corresponding toxicity. Food Chem. 2020; 327: 127103. PubMed Abstract \| Publisher Full Text\n\nZhao X-H, Wang J: A brief study on the degradation kinetics of seven organophosphorus pesticides in skimmed milk cultured with Lactobacillus spp. at 42°C. Food Chem. 2012; 131(1): 300–304. Publisher Full Text\n\nShen C, et al.: Pb(2+) and Hg(2+) removal from polluted milk by di-acrylated Pluronic P123 hydrogels. Food Chem. 2018; 258: 331–336. PubMed Abstract \| Publisher Full Text\n\nMassoud R, et al.: Lead bioremoval from milk by Saccharomyces cerevisiae. Biocatal. Agric. Biotechnol. 2019; 22: 101437. Publisher Full Text\n\nMassoud R, et al.: Lead and cadmium biosorption from milk by Lactobacillus acidophilus ATCC 4356. Food Sci. Nutr. 2020; 8(10): 5284–5291. PubMed Abstract \| Publisher Full Text\n\nMassoud R, et al.: Mercury Biodecontamination from Milk by using L. acidophilus ATCC 4356. J. Pure Appl. Microbiol. 2020; 14(4): 2313–2321. Publisher Full Text\n\nMassoud R, et al.: Mercury biosorption process by using Saccharomyces cerevisiae in milk. J. Food Process. Preserv. 2021; 45(1): 1–9.\n\nMassoud R, et al.: Cadmium Bioremoval by Saccharomyces cerevisiae in Milk. Journal of Medical Microbiology and Infectious Diseases. 2020; 8(1): 29–33. Publisher Full Text\n\nMassoud R, et al.: The Biosorption Capacity of Saccharomyces Cerevisiae for Cadmium in Milk. Dairy. 2020; 1(2): 169–176. Publisher Full Text\n\nAlsager OA, et al.: Removal of antibiotics from water and waste milk by ozonation: kinetics, byproducts, and antimicrobial activity. Ecotoxicol. Environ. Saf. 2018; 158: 114–122. PubMed Abstract \| Publisher Full Text\n\nKitazono Y, et al.: Antibiotic removal from waste milk by electrochemical process: degradation characteristics in concentrated organic solution. J. Mater. Cycles Waste Manag. 2016; 19(3): 1261–1269. Publisher Full Text\n\nKitazono Y, et al.: Selective degradation of tetracycline antibiotics present in raw milk by electrochemical method. J. Hazard. Mater. 2012; 243: 112–116. PubMed Abstract \| Publisher Full Text\n\nAguilar JFF, et al.: Selective removal of tetracycline residue in milk samples using a molecularly imprinted polymer. J. Polym. Res. 2020; 27(7). Publisher Full Text\n\nKumar S, et al.: Photocatalytic, fluorescent BiPO4@Graphene oxide based magnetic molecularly imprinted polymer for detection, removal and degradation of ciprofloxacin. Mater. Sci. Eng. C Mater. Biol. Appl. 2020; 111: 110777. PubMed Abstract \| Publisher Full Text\n\nAlsager OA, Alnajrani MN, Alhazzaa O: Decomposition of antibiotics by gamma irradiation: Kinetics, antimicrobial activity, and real application in food matrices. Chem. Eng. J. 2018; 338: 548–556. Publisher Full Text\n\nBodbodak S, et al.: Selective decontamination of aflatoxin M1in milk by molecularly imprinted polymer coated on the surface of stainless steel plate. Int. J. Dairy Technol. 2018; 71(4): 868–878. Publisher Full Text\n\nIsmail A, et al.: Effect of different microbial concentrations on binding of aflatoxin M 1 and stability testing. Food Control. 2017; 73: 492–496. Publisher Full Text\n\nMoussa AI, et al.: Efficacy of Kaolin and Bentonite Clay to Reduce Aflatoxin M1 Content in Contaminated Milk and Effects on Milk Quality. Pak. Vet. J. 2020; 40(2): 181–186. Publisher Full Text\n\nAbdelmotilib N, et al.: Aflatoxin M1 Reduction in Milk by a Novel Combination of Probiotic Bacterial and Yeast Strains. European J. Nutr. Food Saf. 2018; 8: 83–99. Publisher Full Text\n\nMartinez MP, et al.: Probiotic bacteria and yeasts adsorb aflatoxin M1 in milk and degrade it to less toxic AFM1-metabolites. Toxicon. 2019; 172: 1–7. PubMed Abstract \| Publisher Full Text\n\nAssaf JC, et al.: A novel method for elimination of aflatoxin M1 in milk using Lactobacillus rhamnosus GG biofilm. Int. J. Dairy Technol. 2019; 72(2): 248–256. Publisher Full Text\n\nCarraro A, et al.: Clay minerals as adsorbents of aflatoxin M1 from contaminated milk and effects on milk quality. Appl. Clay Sci. 2014; 88-89: 92–99. Publisher Full Text" }	[ { "id": "121005", "date": "02 Feb 2022", "name": "Pawel Konieczynski", "expertise": [ "Reviewer Expertise analytical chemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn my opinion the article entitled: \"Contaminants in the cow's milk we consume? Pasteurization and other technologies in the elimination of contaminants\" is valuable and covers an important problem from the point of view of studies of food safety, especially milk. It was written based on wide literature screening (about 250 references) and presents the data and conclusions in a clear and comprehensive way. However, I have two comments:\n1. Why didn't the authors discuss the contaminants in milk (metals especially) in the context of norms of PTWI or ADI?\n2. Please add \"Metals and metalloids\" in the heading of Table 2 (page 8) since selenium is included in it.\nMy conclusion: accept after minor revision\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": "121006", "date": "07 Feb 2022", "name": "Esmeralda García Díaz", "expertise": [ "Reviewer Expertise Adsorption and photocatalysis of emerging pollutants of water. Chromatography of compounds of ambiental and biological interest" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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: \"Contaminants in the cow's milk we consume? Pasteurization and other technologies in the elimination of contaminants\" is valuable and presents an exhaustive review of the different types of contaminants in raw/pasteurized cow's milk and analyze the application of alternative processes for the elimination or degradation of contaminants. It provides relevant information about the sources of contamination and the health implications of ingesting these contaminants through milk, adequately supported by citations. It presents a wide variety of contaminants with their details regarding the contamination of milk. Information about alternative treatments to remove contaminants is also relevant and abundant. However, the tables need to be improved to present relevant information, which the authors can surely provide. Please see the following comments regarding the tables:\nCOMMENTS in Figure 1, the \"processing and packaging stages\" label is missing. Plasticizers that are used in containers, such as BPA need to be included. Robert Frankowski, Tomasz Grześkowiak, Beata Czarczyńska-Goślińska & Agnieszka Zgoła-Grześkowiak (2022) Occurrence and dietary risk of bisphenols and parabens in raw and processed cow’s milk, Food Additives & Contaminants: Part A, 39:1, 116-129, DOI: 10.1080/19440049.2021.1986234\nA better title for figure 2 would be \"Incidence of contaminants in bovine...\nPage 4, sentence “For better analysis and understanding, the classification of contaminants according to the origin is microbial contaminants and chemical contaminants (Figure 2)”, needs to be rewritten, since the word \"contaminants\" is repeated several times. I suggest: Figure 2 presents the classification of cow´s milk contaminants and their microbial or chemical origin.\nTable 1 is long but contains relatively little information for its size. The column “Type of milk” is repetitive - separate horizontally by type of milk and add a column with the main identification method used in each case.\nTable 2 is also long and contain repetitive information, with columns labelled as MRL being practically empty. it would be more useful to put the concentration interval reported in the referred works. MRL data can be mentioned in text.\nThroughout the document, round percentage values to make analysis easy.\nPage 14, In the sentence “Therefore, it is important to control the presence of antibiotic residues in food to avoid the appearance of resistance to these antibiotics in humans”. Who becomes resistant, humans or microorganisms? Its unclear in the sentence with the expression “appearance of resistance”. In sentence “Considering that the use of antibiotics in cattle generates residues in milk, their excessive use should be avoided, and the elimination times before milking should be respected in order to avoid the presence of these contaminants”. Please, reference the “elimination times before milking” to support the establishment of this time in some reported work.\nPage 16 The sentence “The literature mentions alternatives for eliminating specific microbial and chemical contaminants”, change 'mentions' to 'reports' instead. This sentence is confusing: “UV-C lamp wattage did not significantly influence the fat concentration in the milk, which means that it is necessary to improve the method to guarantee an effective reduction of these microorganisms if milk transport time exceeds two hours without cooling”. It’s not clear the relationship between lamp wattage, fat concentration, microorganisms and time of cooling.\nMy conclusion: accept after minor revision.\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": "121007", "date": "09 Feb 2022", "name": "Qiansheng Huang", "expertise": [ "Reviewer Expertise public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis review attempts to summarize the knowledge generated with respect to the various types of contamination that cow's milk has and the effect they have on human health. As a central axis, it focuses on strategies to eliminate these contaminants so that their consumption is safer over the population in general. The treated theme is significant, with a real involvement in food security. I consider that the manuscript deserves to be published, not before taking into account the comments detailed below:\nCOMMENT 1: This idea \"they have not demonstrated efficacy in elimination contaminants\" is repetitive in the summary.\nCOMMENT 2: The first paragraph of the introduction is not really a paragraph but a single long sentence. Please correct those errors throughout the document for readability.\nCOMMENT 3: Second paragraph of the introduction, last line: please be specific when \"United States\" e.g. United States of America.\nCOMMENT 4: The last sentence of the first paragraph of section 2, this must be better organized, the phrase is very long and can lead to confusion. Additionally, use the punctuation signs correctly.\nCOMMENT 5: In the fourth paragraph of section 1, reference is made to pasteurization is a method considered for the elimination of \"non-chemical compounds\". Specify what types and metabolites resulting from the application of this process.\nCOMMENT 6: The following phrase, \"For better analysis and understanding, the classification of contaminants according to the origin is microbial contaminants and chemical contaminants (Figure 2)\" must be incorporated into a paragraph, it is not correct to leave it alone.\nCOMMENT 7: In the paragraph before point 2.1, two contiguous phrases initiate in this same way: \"Among chemical contaminants.....\". Please correct that.\nCOMMENT 8: This phrase \"The main types of microorganisms present in milk are bacteria, yeasts, and molds, which represent the different types of microorganisms present in cow's milk.\" It is redundant. Please improve it.\nCOMMENT 9: When a bibliographic reference is made within the text, indicate the year after appointing the authors. Apply in all cases.\nCOMMENT 10: In reference to Figure 1: specify contaminants that come from milk containers (each type).\nCOMMENT 11: Last paragraph of section \"2.\", third line: indicate the reasons why there may be remanence of microbes even after pasteurization, considering that the process (pasteurization) has been carried out correctly.\nCOMMENT 12: SECTION 2.1: Here the \"non-pathogenic\" organisms should be included and those that cause alteration of milk, whose result is harmful to consumer. Improve the format of the tables.\nCOMMENT 13: SECTION 2.2.1, Literal \"ii\". What mean \"According to the analysis of Table 2, Claborn et al.181 report……\". What type of analysis was made with \"Table 2\"?, Who made it?\nCOMMENT 14: SECTION 2.2.1, Literal \"II\". In reference to \"This confirms that skin contaminated with these pesticides is another route of contamination of raw cow's milk.\" The evidence presented is causal, the most appropriate term, in this case, would be \"evidence suggests...\". \"\nCOMMENT 15: SECTION 2.2.1, Literal \"II\". \"In forage...\" is indicated. Specify the conditions of the sample and the possible source of contamination, allowing each case to understand more dynamically. Apply in all cases.\nCOMMENT 16: Throughout the text, there are many redundant ideas in the same phrase or paragraph, please correct that.\nCOMMENT 17: SECTION 2.2.1: Include contaminants such as PFAAs and other organic compounds.\nCOMMENT 18: SECTION 2.2.2: Is not informative and redundant in front of other sections of the document. Improve it by including figures or tables with broader data.\nCOMMENT 19: Linking words and phrases like \"such as....\" are used excessively. Please vary the expressions used in the text. Apply in all cases.\nCOMMENT 20: SECTION 2.2.3, first sentence. Be more specific in the statement.\nCOMMENT 21: SECTION 2.2.3, Second paragraph, first sentence. Here is specified about mastitis, although this pathology has already been named previously. Any specification of some pathology must be made it the first time in which it is named. A similar case occurs with the pasteurization reference on page 16.\nCOMMENT 22: The document has good information, but this is shown a little messy. The writing and sequential logical structure of the manuscript are the main problems. Also, it is necessary to improve the format of the tables. Improve the resolution of Figure 2.\nCOMMENT 23: Specify the harmful effects of each type of contaminant. Specify the accumulation of every contaminant depending on the milk class (which type of cow produces). Specify the difference, in relation to the presence of contaminants, according to the fat content of the milk (whole, half- skimmed, and skimmed).\nCOMMENT 24: SECTION 2.2.5: Include the explanation about the hormones of the cows depending on the life stage of them, and its effect on the consumer.\nCOMMENT 25: SECTION 3: It is messy, it does not have a \"friendly\" order for the reader. Address the actual effect that the pasteurization process has on the structure of each type of contaminant (chemical contaminants).\nCOMMENT 26: TABLE 3: Describe the metabolites produced by alternative methods for the elimination of chemical contaminants.\nCOMMENT 27: Describe the effect of each type of contaminant removal method on the nutritional profile of milk.\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": "121010", "date": "09 Feb 2022", "name": "Lourdes Casas-Cardoso", "expertise": [ "Reviewer Expertise natural products", "supercritical fluid extraction", "countercurrent extraction column", "supercritical impregnation", "active compounds" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 topic: \"Contaminants in cow's milk, pasteurization and alternative technologies in the removal of these contaminants\" is discussed extensively in the context of the paper.\nTable 1 shows the pathogens in cow's milk reported in the literature and Table 2 summarizes the chemical contaminants in cow's milk reported in the literature. All statements are adequately supported by citations.\nPasteurization is a technology used in the industry, however it is exclusive for the elimination of microbial contaminants. Table 3 describes alternative methods to pasteurization: supercritical carbon dioxide, inactivation by pulsed electric fields, inactivation by ozonation. Alternative methods have proven to be efficient in degrading several contaminants; however, they are still not sufficient nor applied on an industrial scale.\nThe conclusions are appropriate. It is suggested to continue research on alternative methods.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-91
https://f1000research.com/articles/10-643/v1	23 Jul 21	{ "type": "Research Article", "title": "Effect of obesity on risk and severity of periodontitis: a cross-sectional study", "authors": [ "Chaerita Maulani", "Elza Ibrahim Auerkari", "Sri Lelyati C. Masulili", "Lindawati S. Kusdhany", "Chiquita Prahasanti", "Nurtami Soedarsono", "Chaerita Maulani", "Sri Lelyati C. Masulili", "Lindawati S. Kusdhany", "Chiquita Prahasanti", "Nurtami Soedarsono" ], "abstract": "Background: The present study aimed to investigate the correlation between obesity and periodontitis, among other risk factors for periodontitis.\n\nMethods: In total, 262 Indonesian male and female subjects were analysed for body mass index (BMI), oral hygiene, plaque index, and clinically evaluated periodontitis. Statistical analysis was performed using Spearman tests and Pearson chi-square tests to estimate the correlation between BMI and periodontitis. Multivariate binary logistic analysis was conducted between covariate and periodontitis. P<0.05 was considered as statistically significant.\n\nResults: The prevalence of obesity was 48.47%. There were positive correlations between BMI and periodontal status for healthy-mild periodontitis, moderate, and severe periodontitis respectively. BMI and periodontitis crude odds ratio (OR) = 2.31 (95% CI 1.41-3.78); p < 0.05, adjusted OR of BMI among other variables, was 1.88 (95%CI 1.05-3.37); p < 0.05. Exploration of the ROC curve found a BMI cut off point of 24.785 kg/m2. Conclusion: Obesity by BMI measurement of ≥ 25kg/m2 correlated to a higher risk of acquiring periodontitis compared to normal-weight individuals.", "keywords": [ "Body mass index", "obesity", "periodontitis" ], "content": "Abbreviations\n\nAUC: Area under curve\n\nBMI: Body mass index\n\nBOP: Bleeding on probing\n\nCAL: Clinical attachment loss\n\nCI: Confidence of interval\n\nCOPD: Chronic obstructive pulmonary disease\n\nIQR: Interquartile range\n\nOHI: Oral hygiene index\n\nOR: Odds ratio\n\nPD: Pocket Depth\n\nPI: Plaque index\n\nROC: Receiver operating characteristic\n\n\nIntroduction\n\nChronic periodontitis shares many common risk factors with other chronic diseases such as hypertension, cardiovascular disease, and type 2 diabetes mellitus. Common risk factors include smoking, alcohol consumption, and obesity. The underlying mechanisms related to obesity are thought to contribute to inflammation and thereby to chronic disease.1\n\nThe etiology of chronic periodontitis derives from the series of complex interactions between pathologic microorganisms in the bacterial plaque and the host and modifications by systemic and local factors.2 Susceptibility to periodontitis also affected by genetic factors which modulate individual responses to the environment and variations of the immune response.3 There are many proinflammatory cytokines involved in periodontitis subjects, such as IL-1, IL-6, TNF-α which also correlates with proinflammatory cytokines in obese patients4 (Makki, 2013). Obesity modulates the host immune response by secreting several proinflammatory factors deriving from adipose tissue (adipocytes) which results in increased bone loss.5\n\nObesity has become a worldwide concern because it’s incidence has nearly tripled since 1975. In 2016 more than 1.9 billion adults 18 years and older were overweight, with 650 millions of those being obese.6 The prevalence of obesity among adults over 18 years old in the U.S. was 39.8% according to a National Health and Nutrition Examination Survey conducted in 2015-2016. Asian non-Hispanic adults had the lowest prevalence (12.7%) compare with all other races and Hispanic-origin groups.7\n\nIn Indonesia according to Indonesian basic health research 2018 (Riskesdas, 2018) the prevalence of obesity in Indonesian adults over 18 years old was 21.8% and the prevalence of periodontitis was 74.1%. Jakarta, the Indonesian capital city, has the second highest obesity prevalence among 34 other provinces at 29,8% whilst the prevalence of periodontitis in urban areas was 71.5%.8\n\nObesity is usually defined by body mass index (BMI) measurement. According to the WHO, ‘overweight’ classification is set at the value of 25.0-29.9 kg/m2 and ‘obesity’ defined as a BMI ≥ 30.0 kg/m2. However in Asian regions BMIs can be set lower than the existing WHO cut-off point, with the overweight BMI at 22-25 kg/m2 and the obesity BMI at 26-31 kg/m2.9 The WHO proposed classification of a weight by BMI in adult Asians for which obesity 1 is defined at 25-29.9 kg/m2 and obesity 2 at ≥ 30 kg/m2.10\n\nIncreased BMI may be a potential risk factor for developing periodontitis.11–13 On the other hand, other studies have shown obesity to be not related to the severity of periodontal disease,14 but associated with tooth loss, oral hygiene and education level.15 Obesity is related to adipokines that are secreted from adipose tissue which has an important role in regulating metabolic and vascular biology.16 Meanwhile, as a result of chronic inflammatory state insulin resistance develops as well as oxidative stress. These factors could be implicated in the possible obesity and periodontitis association.17 BMI and obesity in non-surgical periodontal therapy also appear to be independent predictors of poor response to the treatment.18\n\nThe aim of this study was to ascertain the odds of having periodontitis in obese individuals among other periodontitis risk factors. We hypothesized that obesity correlated with having higher risk of periodontitis compared to normal-weight individuals.\n\n\nMethods\n\nThis cross-sectional study was conducted three subdistrict populations in Central Jakarta from July 2018 to March 2019. The subjects were recruited by consecutive sampling. The inclusion criteria were: healthy male and female participants aged 18-66 years old, having at least 14 teeth, who were willing to participate in the study and sign an informed consent. Exclusion criteria were any disease that could affect the general and oral health of the subject.\n\nOfficial permission was obtained from the corresponding authorities in DKI Jakarta which give legal permission to do the study in Central Jakarta subdistrict and ethical approval was obtained from the Dental Research Ethics Committee of the Faculty of Dentistry, Universitas Indonesia with the protocol number 070390418, and ethical approval number 38/Ethical Approval/FKGUI/V/2018.\n\nSubject height was measured in centimetres and weight was assessed by a calibrated mechanical scale in kilograms. BMI was calculated by ratio weight and height squared (BMI calculated as kg/m2 during data processing). Four categories of BMI defined by the WHO in accordance with the Asia-Pacific perspective were BMI <18.5 kg/m2 as underweight, BMI 18-5-22.9 as normal weight, BMI 23.0-24.9 as overweight, BMI 25.0-29.9 as obesity 1, and ≥30 kg/m2 as obesity 2.10\n\nPeriodontal examination was performed using periodontal probe UNC-15. Clinical parameters of oral hygiene were measured by the simplified oral hygiene index (OHI)19 and plaque index (PI).20 Periodontal status was recorded by pocket depth (PD), recession, and clinical attachment loss (CAL) which measured six sites per tooth except for third molars. Measurements were made in millimetres and were rounded to the nearest whole millimetre. Bleeding on probing was recorded with papilla bleeding index (PBI) by Saxer and Muhlemann21 and the number of teeth also recorded.\n\nPatients were also categorized according to periodontal condition where CAL 5 mm and PD 6 mm were cut off measurements between mild and severe periodontitis.22 Severe periodontitis was determined as CAL ≥ 5 mm at more than 18 sites and PD ≥ 6 mm in at least one site. Moderate periodontitis determined as CAL ≥ 5 mm at 9-18 sites and PD ≥ 6 mm at not more than one site. Mild periodontal status (healthy gingiva, gingivitis and mild periodontitis) was determined by CAL ≥ 5 mm at not more than eight sites and no PD ≥ 6 mm. The periodontal measurement was taken by calibrated periodontists. Analysis of inter-examiner reliability for periodontal status and plaque index was performed, and demonstrated good agreement.\n\nThe Statistical Package for the Social Sciences (SPSS version 23.0) was used to process data. If there were missing data, the samples were excluded from the study. The normality test and descriptive statistics were calculated using Shapiro-Wilk or Kolmogorov-Smirnov tests for distribution with BMI as the dependent factor. Normally distributed data were presented as mean and standard deviation, non-normally distributed data presented as medians and interquartile range (IQR), and categorical data as percentages. We analyzed the correlation between confounding factors and periodontal status with BMI in continuous data. The confounding factors and periodontitis in dichotomy was correlated with five categorical BMI. The correlation between continuous data of clinical parameter periodontal and categorical BMI was assessed. The association between healthy and mild periodontitis, moderate periodontitis, periodontitis and BMI were calculated using Kruskal Wallis tests. Dichotomy of periodontal statuses as dependent variables determined the correlation with periodontitis risk-factors including BMI. The confounding factors were controlled by multiple logistic regression. Both significant crude odds ratio and adjusted ratio were calculated to assess influenced independent variables on periodontal status (95% CI). The effect of each independent variable was measured adjusting for all variables in the model; p < 0.05 was accepted as statistically significant. The ROC was also measured to seek the specific BMI cut-off point in periodontitis (binary) within this study. We included all the subject that match the inclusion criteria until minimal number sample were sufficient.\n\n\nResults\n\nA total of 272 subjects were recruited; however, some subjects were not eligible according to the inclusion criteria: such as having fewer than 14 teeth, or age. Missing data were excluded (Figure 1). Therefore, a total of 262 subjects between the ages of 18 and 66 were included in this study. The percentage of female subjects was larger (59.9%) than male subjects (40.1%) as presented in the general profile (Table 1). The age group of 45-54 made up two fifths of the sample size, whereas the fewest subjects were in the 25-34 years age group. Subject’s education status was mostly high school (53.4%) and the primary occupation was housewife (47.3%). The subjects were mostly non-smokers (79.4%) and non-alcohol-consumers (93.5%). 50.8% of subjects had either healthy periodontal scores or mild periodontitis, and the rest had moderate or severe periodontitis.\n\na Spearman test.\n\nb Pearson test.\n\n** significant < 0.01, 2-tailed.\n\n* significant < 0.05, 2-tailed.\n\nThe socio-demographic and BMI analysis showed significant positive correlations for age, sex, hypertension, and periodontal status. Significant negative correlations were shown for occupation and smoking status (coefficient correlation −0.270, p = 0.000; coefficient correlation −0.142, p = 0.021, respectively) (Table 1). Education, alcohol, DM, and plaque index showed no correlations with periodontal status.\n\nThe prevalence of obesity in this study was 48.47% and the highest prevalence was in BMI group 25.0-29.9 kg/m2 (32.44%), then 18.5-22.9 kg/m2 (29.77%). Table 2 presents the correlation between binary socio-demography data, oral hygiene, and BMI as a dependent factor. A significant positive correlation found between periodontitis and age, sex, occupation, and hypertension and while smoking showed a negative correlation. Continuous data of the clinical parameters of periodontitis and BMI is shown in Table 3. PD, CAL, and PBI were found to have significant correlations with BMI.\n\na Spearman tests.\n\nb Pearson tests.\n\nA significant positive correlation found between BMI and periodontal status. There was and association between mild periodontal status and moderate periodontitis, and between mild periodontal status and severe periodontitis were p = 0.03 and p = 0.04 respectively (Figure 2).\n\nBinary periodontal status categorized moderate and severe periodontitis as one group and healthy periodontal and mild periodontitis as another group. Binary periodontal status was then analyzed as a dependent factor towards the various risk factors (Table 4). The multivariate logistic regression analysis was then performed for variables with p < 0.25 (age, sex, smoking, DM, BMI and OHIS). Diabetes mellitus status and smoking had p-values above 0.25, which were p = 0.301 and p = 0.431 respectively, but both were included in the analyses because DM and smoking status are both important risk factors in periodontal classification.\n\nMultivariate logistic regression analysis showed that periodontitis subjects were more likely to have a BMI ≥ 25 kg/m2 (p < 0.001; adjusted odds ratio 1.881; 95% CI: 1.050-3.371). The risk of periodontitis was higher in male compare to female (p < 0.001; adjusted OR 6.852; 95%CI: 2.540-18.481), and increased with OHI. Adjusted OR for smoking showed that nonsmoking subjects were more prone to periodontitis. The ROC analyses from multivariate logistic regression had an area under the curve of 0.78 (sensitivity 71.3%, specificity 61.7%), determining that it was acceptable using this approach to discriminate between those individuals with healthy or mild periodontitis and severe periodontitis.\n\nDespite the fixed value of the Asian cut-off point in BMI, we tried to estimate the cut-off point of BMI in our subjects. Figure 3 shows that the BMI special cut point with the periodontal status, was found in the value of 24.785 kg/m2 (sensitivity 60.5%, specificity 60.9%, AUC 0.608).\n\n\nDiscussion\n\nA previous study (in which the age ranged from 25 to 66 years) showed that the prevalence of periodontitis was greater among subjects with obesity and the increased was BMI proposed as potential risk factor for periodontitis.12 Sources of potential bias were that BMI and not body fat distribution, which can be calculated by waist circumference (WC), was measured. Fat distribution varies between woman and men and between race/ethnic groups.23,24 Participants were taken from three district in central Jakarta which represent mixed ethnic in Indonesia and can be generalized as Indonesian sub population.\n\nThe present study covered a slightly wider range of age from 18 up to 66 years old. The highest group with severe periodontitis was in the 45-54 years age group (46.5%). Indonesian prevalence data for periodontitis also showed the biggest prevalence was in 45-54 years age group (77.85%).8\n\nThe WHO BMI categories define underweight (<18.5 kg/m2), normal weight (18.5-24.9 kg/m2) and overweight (25.0-29.9 kg/m2), and obese (≥30 kg/m2) while in WHO Asia-Pacific the agreed cut-off for overweight, obese I and obese II category are 23.0 kg/m2, 25-29.9 kg/m2 and ≥30 kg/m2 respectively.10 In the Asia-Pacific area, the BMI cut-off for obesity is 25 kg/m2 rather than the WHO cut-off which is 30 kg/m2.25 We have assumed that the BMI classification for the Asia-Pacific region is more suitable for Asian patients.\n\nThe specific cut off BMI in this study of 24.785 kg/m2 (sensitivity 61.1%, specificity 61%, AUC 0.63) shows resemblance to a study by Suvan et al. which has a cut-off point of 24.32 kg/m2 (sensitivity 61.54%, specificity 61.89%).12 Likelihood ratio (LR) in this study was 1.56 (the disease is 1.56 times more likely in individual with the BMI ≥ 24.78 compare to normal weight < 24.78). This exploration by use of the ROC curve confirmed that using WHO Asian-Pacific standard was more suitable in our study rather than BMI 25-29.99 kg/m2 defined for overweight and BMI ≥ 30 kg/m2 for obesity.6 The BMI increased with increasing age and severity of periodontitis. Both the continuous data and the categorical data showed correlations between BMI and periodontitis. This data was in agreement with previous studies among the population aged 28-5511 and among adults aged 18 to 24 years.26 The clinical parameters of periodontitis (PD, CAL, recession and BOP) presented positive correlations with BMI (p < 0.05). This finding was in line with Zimmermann et al. , a German population cohort study which also found an increased deeper periodontal pocket with higher BMI (OR = 1.6, BMI increase by 5).27\n\nIn this study, education level was at the margin of statistical significance (p = 0.053), as in a previous study which showed association with education, tooth loss, and oral hygiene.15 Tooth loss and oral hygiene were not related to increased BMI in this study, may be because the lowest remaining teeth was 14 teeth as an inclusion criteria while in that study, there was no particular inclusion criteria based on tooth loss. Song et al. in their study found that tooth loss was considered a potential risk indicator for being underweight in Korean adults.28 Regarding poor periodontal health with obesity, subjects had poor compliance towards oral hygiene,13 and obese patients had an approximately three fold higher number of bacterial species present compared with normal weight controls with total of 23 species.27 In this study, oral hygiene was not related to BMI although oral hygiene is significantly related with the severity of periodontitis.\n\nAlthough many studies have shown the relationship between BMI and periodontitis17,27,29,30 as in this study, there are some studies which have on the contrary showed no difference between no or mild periodontitis, moderate periodontitis and severe periodontitis.15,31 Also in another study of BMI and periodontitis in postmenopausal women, those with higher BMI had decreased odds (OR) for having periodontitis compared to participants with normal weight (OR: 0.54; 95% CI: 0.27-0.87) although the obese presented significantly higher clinical attachment loss and gingival index compared to normal and overweight subjects (p < 0.01).32 On the contrary, Puspitadewi et al. in the study of postmenopausal women found no significant correlation between age, BMI, bone density and alveolar bone resorption (p > 0.05).33\n\nFemales had a higher BMI compared to male subjects, and according to occupation housewives have the highest BMI compared to employed subjects and students, this corresponds with other studies that have found females to have a higher BMI compared to males.15 Smoking subjects in this study had a lower BMI than non-smoking subjects. Smoking was not correlated with periodontitis, but after adjusting other covariates, a significant correlation was found between non-smokers and periodontitis. This may be due to the low percentage of smokers (20.6%) compared to non-smokers (79.4%) in our study. BMI has multiple risk factors (Table 2) but after multivariate logistic regression with BMI as dependent factor, only age give a significant result p < 0.001; OR 3.63 (95% CI, 1.86-7.12).\n\nMultivariate regression in role factors in having periodontitis, adjusted OR showed sex, OHI, smoking, and BMI, p < 0.001, OR 6.852 95% CI; p < 0.001, OR 5.189 95% CI 2.752-9.784; p = 0.007, OR 3.803 95% CI 1.449-9.979; and p = 0.034; OR 1.881 95% CI 1.050-3.371 respectively. Together, it means obese male non-smokers with bad oral hygiene are prone to periodontitis.\n\nThe odds ratio for BMI was 1.881 (95% CI 1.050-3.371) slightly higher than a previous study where OR was 1.56 (95% CI 1.26-1.92).26 This finding was contrary to a study in Korea by Kim et al. who with multivariate analysis found no association between BMI and periodontitis with BMI ≥ 25, adjusted ratio 0.991 (95% CI 0.806-1.220) but found a significant association between abdominal obesity and periodontitis with an adjusted odds ratio of 1.358 (95% CI 1.003-1.839).14 Associations between obesity and periodontitis were found more consistently for visceral than general adiposity, suggesting that visceral fat accumulation measurement may be more strongly associated with periodontitis more than BMI.29 So we suggest for further study including waist hip ratio as a comparing method for BMI measurement.\n\n\nConclusion\n\nThis study showed a significant correlation between BMI and periodontitis in Indonesian adults. Therefore, BMI evaluation can be used as a factor for assessing the risk of periodontitis.\n\n\nData availability\n\nHarvard Dataverse: Raw Data of effect of obesity on risk and severity of periodontitis. https://doi.org/10.7910/DVN/MBVN3O.33\n\nThis project contains the following extended data:\n\n• Data_Subject_ObesityResearch.tab (raw per subject data).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nWritten informed consent for publication of the patient details was obtained from the patients.\n\n\nAuthor contributions\n\nCM: Data Curation, Investigation, Visualization, Writing-Original Draft Preparation, Writing-Review & Editing; EIA: Conceptualization, Recourses, Supervision, Validation, Writing-Review & Editing; SLM: Methodology, Recourses, Supervision; LSK: Formal Analysis, Supervision, Validation; CP: Formal Analysis, Supervision; NS: Data Curation, Formal Analysis.", "appendix": "Acknowledgments\n\nThe authors wish to acknowledge the Universitas Indonesia for funding the work.\n\n\nReferences\n\nReynolds M: Modifiable Risk Factors in Periodontitis: At the Intersection of Aging and Disease. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhardwaj VK, Sharma D, Jhingta P, et al.: Assessment of Relationship between Body Mass Index and Periodontal Status among State Government Employees in Shimla, Himachal Pradesh. J Int Soc Prev Community Dent. 2013; 3(2): 77–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimmermann H, Hagenfeld D, Diercke K, et al.: Pocket Depth and Bleeding on Probing and Their Associations with Dental, Lifestyle, Socioeconomic and Blood Variables: A Cross-Sectional, Multicenter Feasibility Study of the German National Cohort. BMC Oral Health. 2015; 15: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSong IS, Han K, Ryu JJ, et al.: Association between Underweight and Tooth Loss among Korean Adults. Sci Rep. 2017; 7: 41524. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeller A, Rohde JF, Raymond K, et al.: Association between Periodontal Disease and Overweight and Obesity: A Systematic Review. J Periodontol. 2015; 86(6): 766–76. PubMed Abstract \| Publisher Full Text\n\nSuvan JE, Finer N, D'Aiuto F: Periodontal Complications with Obesity. Periodontol 2000. 2018; 78(1): 98–128. PubMed Abstract \| Publisher Full Text\n\nSusanto H, Nesse W, Kertia N, et al.: Prevalence and Severity of Periodontitis in Indonesian Patients with Rheumatoid Arthritis. J Periodontol. 2013; 84(8): 1067–74. PubMed Abstract \| Publisher Full Text\n\nAl Habashneh R, Azar W, Shaweesh A, et al.: The Relationship between Body Mass Index and Periodontitis among Postmenopausal Women. Obes Res Clin Pract. 2016; 10(1): 15–23. PubMed Abstract \| Publisher Full Text" }	[ { "id": "90953", "date": "13 Aug 2021", "name": "Euis Reni Yuslianti", "expertise": [ "Reviewer Expertise Bochemistry and Biomolecular", "Oral Biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this paper excellent and is an important addition to the literature.\nPlease provide more depth exploring in the discussion, first paragraph – as noted before about the correlation between socio-demographic data, oral hygiene and continuous data of body mass index including alcohol, DM, and plaque index.\nPlease provide more literature and a deeper discussion about how obesity can effect on risk and severity of periodontitis.\nLinking to the results, please include more detail about the correlation between BMI, obesity, and metabolic syndrome to severity of periodontitis.\nPlease add more depth exploring in the discussion related to adipokines that are secreted from fat cells (adipocytes) in obesity which has an important role in regulating metabolic and oxidative stress to periodontitis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "7639", "date": "25 Jan 2022", "name": "Elza Ibrahim Auerkari", "role": "Author Response", "response": "Thank you so much for your kind suggestions for our article. Regarding your comment we completed as follows: the aspects of discussion in the first paragraph, have been addressed in the revised paper. More literature and a deeper discussion about how obesity can effect the severity of periodontitis also have been added to the discussion and references of the revised paper. More detail about the correlation between BMI, obesity, metabolic syndrome to the severity of periodontitis, and discussion about adipokines role in regulating metabolic and oxidative stress to periodontitis have been added to the discussion of the revised paper." } ] }, { "id": "90954", "date": "15 Sep 2021", "name": "Muhammad S. Zafar", "expertise": [ "Reviewer Expertise Dentistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title of the study is clear and concise.\n\nThe abstract of the study is adequately written, mentioning the key aspects of the paper.\n\nThe introduction is clearly written with relevant and latest citations. However, the authors should mention the names of proinflammatory cytokines before abbreviating them (Page 3, second paragraph). Furthermore, the introduction should be expanded a bit as it seems to be brief, especially the first and second paragraphs.\n\nIn the methods section, the authors should mention the STROBE checklist as it is an observational study.\n\nIn the methods section, the authors should separate the diagnostic criteria of periodontal disease and obesity by giving them separate sub-headings.\n\nThe authors should also mention the null hypothesis of this study.\n\nThe authors should carefully check the use of abbreviations in the main text, tables, and figures.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7640", "date": "25 Jan 2022", "name": "Elza Ibrahim Auerkari", "role": "Author Response", "response": "Dear Sir, thank you so much for the constructive suggestion in our articles. Hereby we revised the article accordingly. The suggested changes and extensions have been implemented in the introduction, paragraphs 1 and 2, of the revised paper. The strobe checklist has been added in the methods section. also the separate the diagnostic criteria between periodontal disease and obesity by giving them separate sub-headings. The null hypothesis of this study has been added to the methods section and the use of abbreviations has been checked and missing ones added." } ] } ]	1	https://f1000research.com/articles/10-643
https://f1000research.com/articles/11-87/v1	24 Jan 22	{ "type": "Research Article", "title": "The influence of extrovert and introvert personality types on the acceptance of virtual learning during the COVID-19 pandemic: a survey", "authors": [ "Siti Rasyidah Sanudin", "Hawa Rahmat", "Maizatul Azila Chee Din", "Najihah Akeb-Urai", "Siti Rasyidah Sanudin", "Maizatul Azila Chee Din", "Najihah Akeb-Urai" ], "abstract": "Background: As a result of the COVID-19 pandemic, all courses taught in Malaysian schools and universities were conducted entirely virtually, after a movement control order was imposed in Malaysian on 18th March 2020. According to the research by Offir et al., (2007), extroversion-introversion (E-I) personalities have been shown to influence students’ involvement in class, their learning style and their understanding of the course materials. The purpose of this research was to explore how extrovert and introvert personality traits influence the acceptance of virtual learning amid the COVID-19 pandemic. Methods: A case study was conducted, focusing on a group of students taught by one of the researchers in our research team, HR. A total of 31 Diploma students (13 first year students and 18 second year students) taking Grooming and Professional Etiquette at Multimedia University, Malaysia (MMU) participated in the study. Open-ended questions were used to learn about the personality of each participant, as well as to provide a better understanding on how the opposing personas differs in their experiences with virtual learning. Results: Our results showed that 54.83% (17) of the students were introverts, 29.03 % (9) were extroverts, and 16.1% (5) were private-public-introvert-extroverts (PPIEs). The results for the acceptance level of virtual learning varied based on the different personality types. Results showed the extroverts expressed that the virtual learning experience was unpleasant, while the introverts and the PPIEs found virtual learning a useful and pleasant experience. Conclusion: This study was mainly descriptive, with open-ended questions used to gain insight on how different personality types differ in their acceptance of fully virtual learning. In future studies, inferential analysis could be carried out to test the hypotheses and assumptions. Future studies could also compare and contrast both students and lecturers’ acceptance of, and engagement in, online learning during the COVID-19 pandemic.", "keywords": [ "personality", "extroversion", "introversion", "Covid-19", "virtual learning" ], "content": "Introduction\n\nOne of the consequences of the COVID-19 pandemic is that many universities in Malaysia were forced to deliver classes entirely online, due to the movement control order (MCO) imposed in Malaysian on 18th March 2020.1 Online classes mean students are not able to have personal or face-to-face contact with their teachers and other students and are not able to participate in discussions in the same way. Based on students respective personalities, these learning conditions may affect students’ experience of virtual classes differently. Extroversion-introversion (E-I) variations have been shown to affect how students engage in class, their learning style and the way they process knowledge.2,3 Most studies have focused on students and teachers’ engagement in online classes.4 However, very few studies have investigated how the differences in personality affect online learning, especially in relation to the acceptance and perception of virtual classes among students. As of March 2020 university classes are being conducted online, which presents a valuable opportunity to explore this question. The study's original goal was to investigate if there were any relationship between resilience and acceptance of virtual learning classes, across different personality types. However, owing to a lower number of responses from students than expected, the quantitative study was terminated, and instead, a qualitative analysis was conducted on various personalities types and students' acceptance of virtual classes. The study’s research question then evolved into trying to understand whether personality types influence students’ acceptance of virtual classes. Therefore, the objective of this paper is to explore how various personality types influence students’ acceptance of virtual learning classes during the COVID-19 pandemic.\n\nResearch has shown that the psychological and emotional impact of the COVID-19 pandemic has been present from the very beginning. Roy et al. (2020), conducted a study in India using a questionnaire and non-probability snowball sampling technique; the study revealed that those who were concerned about the spread of COVID-19, had higher levels of anxiety.5 This leads us to ask how young adults are coping with the new experience of a fully virtual class load, in addition to anxiety around the pandemic and indicates the need to investigate students’ level of acceptance of virtual learning and their feelings around such experiences.\n\nIn the province of Davao del Sur, southern Philippines, another study conducted in 2020 which focused on students' knowledge, attitude, anxiety, and coping mechanisms during the COVID-19 pandemic found that they were uninterested in the online-blended learning approach.6 In the study, 59.25% (314 out of 530) of the students disagreed with the conduct of the online-blended learning approach. The study revealed that the primary reason for the disagreement was poor internet connection, which was reported by 72.29% (227 out of 314) of the students. However, even though in that study technical problems were the main cause, we believe personality factors could not be ignored since differences in personality may also affect students’ acceptance and perception of online learning.2\n\nExtroverts are described as having a diverse range of interests, being proactive in their work and relationships, are sociable, and have a strong sense of self-expression. According to research, extroverts enjoy events such as classroom discussions, group projects, and opportunities to engage with the teachers, and brainstorming or discussing ideas.3,7\n\nIntroverts tend to engage in activities such as listening and reflection before responding to questions from the teachers and prefer writing as a means of communication and contemplation as a means of working out ideas.3 Generally, this is also their preferred way of learning and they perform best when given time to reflect and process before participating in classroom activities, while extroverts need a high degree of stimulus to stay engaged. Research has reported that various strategies have been used by students to cope with learning during the pandemic such as reminding themselves of the positive benefits it has in limiting the spread of the virus and protecting them from exposure.6 Respondents who avoided thinking about the pandemic or those struggling to cope, had significantly greater anxiety and depression.6\n\nA PPIE individual is in-between an extrovert and introvert. PPIEs are also known as ambiverts.8 This type of personality will become agile, active, and energetic when they are within their small circle of friends or when they are with their own family.9 However, when they are with a stranger, they are quiet and seem less interested to start a conversation.10 Zholudeva et al. (2021) also found that ambiverts or PPIEs had a greater degree of preparedness for a future career, than extroverts and introverts, when looking at the readiness of students for professional careers.11\n\n\nMethods\n\nWritten informed consent was obtained from the respondents, for both involvement in the study and use of data and students could also withdraw from participating in the survey. Ethical approval was granted by Research Ethics Committee of the Technology Transfer Office, Multimedia University, Malaysia. Ethical approval number: EA0712021.\n\nWe conducted a case study on a group of students (n = 31) in one of the classes taught by the second author in the research team, HR. The survey questionnaires were administered to the students via Google Forms during a virtual class12 in Grooming and Professional Etiquette, at Multimedia University, Malaysia on 27th May 2021. This subject is offered for students in their first and second year of their studies. A six item resilience questionnaire was adapted from the Brief Resilience Scale (BRS)13 and included in the survey. A five-item Likert scale, ranging from strongly disagree to strongly agree was also incorporated; including statements aimed to establish students’ resilience, such as “I tend to bounce back quickly after hard times”. Data collected included information about the demographic background of the students such as gender, religion, ethnicity, year of study, and personality traits. The students were given a link to a personality test website specifically designed for identifying their personality traits.14 The students took the test online and results were automatically generated. There are three types of personality generated: extroverts, introverts, and PPIEs. Then students were encouraged to ask questions regarding the survey, if they did not understand any of the parts. Finally, the students were instructed to submit the Google form once they have completed the survey. The analysis of students’ acceptance of online learning was carried out based on the open-ended questions which were asked, to gain insight on the students’ experience, acceptance and perception of the virtual learning experience. The questions were as follows (see Extended data for a copy of the survey12):\n\n- Do you like the classes to be conducted 100% online? Why and why not?\n\n- Do you prefer 100% online classes or would you prefer a certain percentage of classes be face to face? Why and why not?\n\n- When the COVID-19 pandemic is over, do you still want online classes for this subject?\n\n\nResults\n\nThe responses from the students did not meet the sample size recommendation by Krejcie and Morgan.15 Thus, due to a lower than the projected number of responses from students, the resilience questions are excluded in the analysis, and instead, a qualitative analysis on various personality types and students' acceptance of virtual classrooms was done. A total of 31 students participated in the study. The demographic background of the student participants is shown in Table 1.\n\nThe findings obtained from this study revealed interesting differences between extrovert and introvert students in relation to their acceptance of virtual learning. Table 2 showed the results of personality types and the acceptance of virtual learning. Based on the test that the students took, 55% (17) of the students were categorised as introverts, 29 % (9) were extroverts, and 16% (5) met the private-public-introvert-extrovert personality type. About 61% (19) students positively accepted virtual learning and 39% (12) students did not enjoy virtual learning during the pandemic. From the 19 students who positively accepted virtual learning during the pandemic, 58% (11) of them were introverts, 32% (6) extroverts, and 10% (2) PPIEs.\n\nThe results reveal that the introverts seemed happy attending fully virtual classes and preferred staying at home rather than meeting people. This is consistent with the characteristics of introverts; being people who do not like to be around large groups and can tend to avoid public places or crowded events.10 Introverts also prefer to communicate through writing.\n\nCompared to extroverts, introverts perform best when given time to reflect and process before participating in classroom activities.7 The results of our study confirmed the findings of research conducted by Offir et al., (2007), in which extroverts were found to dislike virtual classes.2 For the extroverts in our study, having a certain percentage of classes conducted online is acceptable; however, they could not completely focus if they had to attend fully virtual classes. They preferred meeting people, talking to friends, and consulting their lecturers face-to-face. This was evidently expressed by one of the extrovert first-year female students (respondent no. 17), who mentioned in the open-ended question that “I must see people, meet people, talk to real people. I need a high degree of stimulus to stay involved”.12\n\nWe found that PPIE students did not like virtual classes, but that they are quite tolerant of such classes because of the situation created by the pandemic. Nevertheless, they claimed that they have to contact friends and lecturers after classes, for further help to understand the topic being studied. PPIE students reported that the virtual classes are satisfying enough if they have at least one or two classes where they are able to meet up in person with other students or teachers. This is in-keeping with the traits of PPIEs, whereby being able to meet and talk to people in person every now and then, is sufficient.\n\nExtroverts are described by C. G. Jung (1971), who is a key figure in the study of personalities, as being oriented primarily toward the outside world, and as such, they prefer to focus their observation and judgment on people and objects. Introverts, on the other hand, are more focused on the inner world and thus, prefer to focus their observation and judgment on thoughts and ideas.16 As such, in any online class, there will likely be introvert students paying close attention and focusing on what the lecturer is trying to convey and working hard to complete all the work assigned to them. The extroverts might find being confined to one place, within which they are not able express their concern to the lecturer or fellow students, annoying and stressful.10 The findings of our study suggest that online learning is valuable, but a certain percentage of face-to-face activities are needed for a better student-teacher and student-peer engagement. Furthermore, according to Lestari et al. (2013), introvert and extrovert students have different learning styles; introvert students preferring to study alone while extroverts prefer to study in groups.7\n\n\nConclusion\n\nThe findings indicate that the students are aware of and understand the need for online learning during the COVID-19 pandemic. They have no choice but to follow the new norm of learning in the current situation. For now, online learning is the only way that teacher-student interaction can be conducted in some countries, and one must adapt to the changing environment.\n\nThe results of the open-ended questions help researchers better understand how extroverts, introverts, and PPIE students perceive and embrace completely virtual learning programs. Even though the introverts indicated that virtual classes were ok for them, they stated that they still needed to see people in person to keep them “alive” and “intact”.12 The urge to meet others, such as friends, classmates, and lecturers, is for study objectives rather than social purposes for introverts. Extroverts must interact with actual people in order to communicate, be heard and listen and love sharing their lives with others; not just for academic objectives.\n\nArguably, online learning, in a more structured way, could be the future of our teaching and learning. Perhaps, what we are facing now is just the beginning of a more complicated but perhaps stimulating future of education. However, the study cannot be generalised to all Malaysian students since it was only a case study with a small group of students in a Grooming and Professional Etiquette class at Multimedia University. Further research is therefore required to replicate this study on a larger scale, to aid better generalisability. More research is suggested to understand further the differences in personality and how it affects perceptions of online learning, with the hope that such research would be able to inform and improve teaching and learning methods in the future.\n\n\nData availability\n\nDANS: The influence of extroversion and introversion personality types on the acceptance of virtual learning during the COVID-19 pandemic, https://doi.org/10.17026/dans-2zc-x4u2.12\n\nThis project contains the following underlying data:\n\n• DATASET_PAPER ONLINE LEARNING AND PERSONALITY.csv\n\nDANS: The influence of extroversion and introversion personality types on the acceptance of virtual learning during the COVID-19 pandemic, https://doi.org/10.17026/dans-2zc-x4u2.12\n\nThis project contains the following extended data:\n\n• F1000_questionnaire_GROOMING CLASS ACCEPTANCE ON VIRTUAL CLASSES - Google Forms.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThank you to the students in Grooming and Professional Etiquette class at Multimedia University, Malaysia that participated in the study.\n\n\nReferences\n\nTang K: Movement control as an effective measure against Covid-19 spread in Malaysia: an overview. Zeitschrift fur Gesundheitswissenschaften = Journal of Public Health. 2020; 1–4. Advance online publication. Publisher Full Text\n\nOffir B, Bezalel R, Barth I: Introverts, extroverts, and achievement in a distance learning environment. Am. J. Dist. Educ. 2007; 21(1): 3–19. Publisher Full Text\n\nMurphy L, Eduljee NB, Croteau K, et al.: Extraversion and introversion personality type and preferred teaching and classroom participation: A pilot study. J. Psychosoc. Res. 2017; 12(2): 437–450.\n\nAguilera-Hermida AP, Quiroga-Garza A, Gómez-Mendoza S, et al.: Comparison of students’ use and acceptance of emergency online learning due to COVID-19 in the USA, Mexico, Peru, and Turkey. Educ. Inf. Technol. 2021; 26: 6823–6845. Publisher Full Text\n\nRoy D, Tripathy S, Kar SK, et al.: Study of knowledge, attitude, anxiety & perceived mental healthcare need in Indian population during COVID-19 pandemic. Asian J. Psychiatr. 2020; 51: 102083. PubMed Abstract \| Publisher Full Text\n\nBaloran ET: Knowledge, Attitudes, Anxiety, and Coping Strategies of Students during COVID-19 Pandemic. J. Loss Trauma. 2020; 25: 635–642. Publisher Full Text\n\nLestari A, Sada C, Suhartono L: Analysis On the Relationship of Extrovert; Introvert Personality and Students Speaking Performance. Jurnal Pendidikan dan Pembelajaran Khatulistiwa. 2013; 4(3).\n\nLuong V, Shields C, Petrie A, et al.: Does Personality Matter? Perceptions and Experiences of Introverts and Extraverts as General Surgeons. Teach. Learn. Med. 2021: 1–11. PubMed Abstract \| Publisher Full Text\n\nDowns GH: An exploration of the relationship between personality type and satisfaction with online learning environments. 2019 Portland International Conference on Management of Engineering and Technology (PICMET). 2019, August; (pp. 1–4). IEEE.\n\nBishop-Clark C, Dietz-Uhler B, Fisher A: The effects of personality type on web-based distance learning. J. Educ. Technol. Syst. 2007; 35(4): 491–506. Publisher Full Text\n\nZholudeva S, Zhukova R, Naumenko M: Readiness for the Profession of Students With Different Psychotypes in a Digital Educational Environment. E3S Web of Conferences 2021; (Vol. 258). EDP Sciences.\n\nSanudin SR: The influence of extroversion and introversion personality types on the acceptance of virtual learning during the COVID-19 pandemic. DANS. 2021. Publisher Full Text\n\nSmith BW, Dalen J, Wiggins K, et al.: The brief resilience scale: assessing the ability to bounce back. Int. J. Behav. Med. 2008; 15(3): 194–200. PubMed Abstract \| Publisher Full Text\n\nBishop-Clark C, Dietz-Uhler B, Fisher A: Test: Are you an introvert or an extrovert?.2018. Retrieved on 30 May 2021. Reference Source\n\nKrejcie RV, Morgan DW: Determining sample size for research activities. Educ. Psychol. Meas. 1970; 30(3): 607–610. Publisher Full Text\n\nJung CG: Psychological types Trans. Baynes HG, Hull RFC, editors. Princeton, NJ: Princeton University Press; 1971. (Orig. pub. 1921.)" }	[ { "id": "120901", "date": "11 Feb 2022", "name": "Nina B Eduljee", "expertise": [ "Reviewer Expertise Educational Psychology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study was interesting and the authors recognized the limitations of the study (n = 31), and quickly pivoted to conducting a descriptive study. It would have been interesting to see what % of males and females were introverts and extroverts. That data can be added easily.\n\nDoes this journal have a word limit on articles? A couple of other studies (or citations if there is a word limit) could be added to make to the literature review more robust.\n\nThe authors state that they excluded the resilience questions due to not meeting sample size recommendations. Did the authors not examine the 9 questions on acceptance of remote learning (maybe mean/SD could be presented)? That can also be added as part of the results easily.\n\nThe open-ended questions were interesting but I was not able to access the responses, so had to go with the narrative the authors provided. Did they look for themes with the data? That could have been included with the results or some statements by the students could have been added in the narrative.\n\nThe conclusion addressed the key issues of the generalizability of the study. It would be very interesting to replicate on a larger scale to see if the results hold.\n\nOverall, an interesting study that can be made even more robust with minor changes and additions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "120905", "date": "23 Feb 2022", "name": "Azlina Mohd Khir", "expertise": [ "Reviewer Expertise Social Psychology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, the presentation of this article is clear, readable and interesting. However, some suggestions could be taken into consideration.\n\nThe abstract is well explained. However, citations should not be used in the abstract.\n\nThe introduction should explain in-depth the virtual learning itself, including its conceptual definition (with citations).\n\nThe literature review is well explained. However, it is suggested to add more citations to make the literature review more robust.\n\nMethods: Please explain the population and sampling technique used in the study. How was the data analyzed? What techniques did you use to analyze the quantitative and qualitative data? Please explain in detail.\n\nResults:\n\nYou need to re-check the data in Table 2. There is a typo for the total number of PPIE respondents. The correct number is 5, not 16 (equivalent to 16%).\n\nThe total number of Introverts is 17, if 11 respondents answer 'Yes' to Acceptance on virtual learning during the pandemic, so the number who answered 'No' will be 6. Why have you stated it is 4? Also for Extroverts, the total number of Extroverts is 9. If 6 respondents answer 'Yes' for the Acceptance of virtual learning during the pandemic, why have are you stated 5 in the ‘No’ column, and not 3? Also, re-check the frequency for Introverts and Extroverts who answer 'No' to the Acceptance of virtual learning after the pandemic. How can Table 2 describe which personality type is more accepted in virtual learning? Highly suggested to add the results from the Acceptance on Remote Learning scale. How do you analyze the open-ended questions? It is suggested to explain well in this section, e.g. based on the themes, or some statements by the respondents that can be added to the narrative.\n\n6. The conclusion is well explained.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-87
https://f1000research.com/articles/11-86/v1	24 Jan 22	{ "type": "Research Article", "title": "Effect of pulsed electromagnetic field versus pulsed high intensity laser in the treatment of men with osteopenia or osteoporosis: a randomized controlled trial", "authors": [ "Anwar Ebid", "Shamekh El-Shamy", "Ali Thabet", "Mohamed El-boshy", "Mohamed Abedalla", "Tariq Ali", "Anwar Ebid", "Ali Thabet", "Mohamed El-boshy", "Mohamed Abedalla", "Tariq Ali" ], "abstract": "Background: Osteoporosis has been related to a negative impact on several aspects of patient health, including physical, mental, and emotional well-being. The objective of this study was to examine the effects of pulsed electromagnetic fields (PEMF) and pulsed Nd-YAG laser therapy (HILT) on men with osteopenia or osteoporosis.\n\nMethods: Ninety-five men with osteopenia or osteoporosis (mean age, 52 years; mean height, 176 cm; mean weight, 83 kg; mean body-mass index (BMI), 26.86 kg/m2) took part in the study, and they were randomly assigned to one of three groups: Group 1 received PEMF and exercise program (PEMF +EX), Group 2 received HILT and exercise program (HILT+EX), and Group 3 received exercise program only (EX). PEMF was applied three times per week for 12 weeks using a full-body mat, while HILT was applied to the lower back and hip regions with a total dose of energy of 3000 J delivered in two treatment stages. Flexibility, aerobic exercise, strength, weight-bearing, and balance exercises are included in exercise program, which is followed by whole-body vibration training. Bone mineral density (BMD) of the total hip and lumbar spine, bone markers, health-related quality of life (HRQoL), and fall risk are all outcome measures. Results: There were no significant differences in the parameters between the groups at the baseline (P > 0.05). Patients in all groups, however, showed significant improvements in all measured parameters following treatment (P< 0.05), with Group 1 and Group 2 showing much greater improvements than Group 3. Conclusion: After 12-weeks of treatment, PEMF combined with exercise is more effective than HILT combined with exercise or exercise alone in increasing BMD and promoting bone formation, suppressing bone-resorption markers, and improving quality of life and fall risk, with the effects lasting up to six months. This study was registered in the ClinicalTrial.gov PRS (NCT05029440, 26/08/2021).", "keywords": [ "Pulsed electromagnetic field", "pulsed high intensity laser", "Exercise program", "bone mineral density", "osteopenia", "osteoporosis." ], "content": "Introduction\n\nOsteoporosis (OP) is a systemic bone disease characterized by a gradual loss of bone mass and tissue degradation, resulting in impaired bone strength, significant deterioration of bone microarchitecture, and decreased bone mass, caused by bone resorption outpacing bone formation, resulting in increased bone fragility and a high fracture risk.1,2 Osteopenia is a condition marked by low bone mineral density (BMD) that can lead to OP. Because patients have no early symptoms, most OP cases are detected after a fracture.3\n\nOsteoporosis and the fractures that accompany it are a major public health concern around the world. OP is estimated to affect over 200 million people worldwide, resulting in over 8.9 million fractures each year.4 OP affects 34% of women and 30.7 % of men aged 50 to 79 years in Saudi Arabia.5\n\nOsteoporosis has been related to a negative impact on several aspects of patient health, including physical, mental, and emotional well-being, with hip and vertebral fractures having the greatest impact on patients' quality of life.6 An increase in fragility fractures is a major complication of OP, which leads to severe morbidity, mortality, disability, and a deterioration in patients' quality of life.7,8\n\nDespite current advancements in healthcare systems, the prevalence of OP among older adults in Saudi Arabia continues to increase, and proven effective medication to treat low BMD and decrease the risk of fractures is becoming more widely available.9 Unfortunately, because OP is asymptomatic, its care is currently inadequate, and the illness is frequently undiagnosed and untreated until it has progressed to the point where fractures occur.10\n\nThere is currently no universally approved strategy for therapeutic decision-making in men. To minimize fracture risk, a daily calcium intake of 1000-1200 mg (from dietary sources or supplements) and adequate vitamin D intake to achieve 25 (OH) D concentrations of >30 ng/ml are recommended.11 Men should participate in regular weight-bearing and muscle-strengthening physical exercise, as well as quit smoking and reduce their alcohol consumption.12\n\nExercise is a well-known method of preventing bone loss as we age, but high-intensity loading is required to trigger a beneficial adaptive response.13 Progressive resistance training combined with weight-bearing impact activity is most likely the most advanced form.14 Closed kinetic chain exercises can help postmenopausal women with OP increase their BMD and decrease their fall risk. As a result, these activities should be used in conjunction with other OP therapy strategies.15\n\nPulsed high-intensity laser therapy (HILT) penetrates the tissue deeply, triggering chemical and mechanical changes as well as thermal mechanisms.16 Physical properties of HILT beams, such as mechanical and thermal effects, may be responsible for the profibrinolytic effects. Inflammation and painful sensations are promptly reduced by HILT.17 It employs a waveform with regular amplitude peaks and shot duration that induce photochemical and photothermic effects in deeper tissues, resulting in increased blood flow, vascular permeability, cell metabolism, and tissue photomechanical level, all while requiring relatively short application times.3\n\nPulsed electromagnetic field (PEMF) has a lot of potential to become a stand-alone or adjuvant treatment approach for musculoskeletal disorders such osteopenia and OP because of its noninvasiveness, safety, and efficacy.18 Several studies have investigated at the underlying cellular and subcellular mechanisms of PEMF stimulation on a variety of musculoskeletal disorders, providing a molecular basis for expanding its therapeutic application.19–22\n\nThe paucity of studies focusing on such a high-impact population in Saudi Arabia is making it difficult to establish effective intervention programs that address specific concerns for this group.23,24 The goal of this research was to compare the effects of PEMF and HILT in the treatment of men with osteopenia or osteoporosis.\n\n\nMethods\n\nThis was a randomized regulated study with three measurement intervals. Umm Al-Qura University's Biomedical Research Ethical Committee accepted and deemed this project properly achievable (Approval number: HAPO-02-K-012-2021-540). The study was also listed with the Clinical Trial Registry (Clinical Trials.gov ID: NCT05029440).\n\nThe proper sample size was calculated using G-power for Windows, with estimated power = 0.95 and = 0.05. The effect size was 0.30 utilizing evaluation of variance (ANOVA) with in-between interface in three groups and three assessment times. A minimum of 92 patients was identified in all therapy groups.\n\nNinety-five men over the age of 45 were enrolled in the study (mean age, 52.02 ± 1.83 years; average height, 176 ± 1.89 cm; mean weight, 83.21 ± 2.34 kg; average BMI, 26.86 ± 1.34 kg/m2). All applicants were initially evaluated for the enrollment requirements. The screening method included a health and psychiatric record, involving height, weight, and BMI, in addition physical and lab analysis. Every participant was physically evaluated to determine their level of physical activity. During the examination, any gait difficulties, muscular distress, or joint discomfort were reported. If applicants had physical treatment or a shift in their pharmacological treatment in the prior three months, they would not be allowed to use any extra treatments, specific regimens, or aerobic training options for the duration of the study.\n\nParticipants were told to approach their standard exercises as regular all through the preliminary and to stay away from any methodical exercise preparing programs. Each of the patients were determined to have osteopenia or osteoporosis (T-scores of <−1.5) utilizing dual-energy X-ray absorptiometry (DEXA). All members were assessed by a similar assessor before treatment, following 12 weeks of treatment, and six months after the end of the treatment. Follow-up was done simultaneously following a half year assessment to control for diurnal varieties. Following the pre-treatment assessment, the review objectives were tended, and all members gave composed informed assent for their interest and the distribution of their review results. The study's stream outline shows the entirety of the proposal means (Figure 1).\n\nDiabetes, intraocular focal points, coagulation record, cardiopulmonary disorders, extreme vascular and kidney sickness, thyroid illness, heart pacemaker, life-threatening blood pressure, advanced neurological manifestation, persistent impairing joint pain, utilization of any drugs that influence bone mineralization, serious liver sicknesses, presence of osteoporotic crack, critical sickliness, neuropsychiatric problems (e.g., dementia), alcohol misuse, extreme sadness, bipolar syndrome, or psychosis and BMI <19 kg/m2 or >31 kg/m2 were completely avoided from the scheme.\n\nThe applicants were arbitrarily allocated to one of three groups after the initial evaluation of using an electronic GraphPad Prism (RRID:SCR 002798; R is an open access alternative). Group 1 received PEMF combined with an exercise program (PEMF+ EX), Group 2 received HILT combined with an exercise program (HILT+ EX), and Group 3 received an exercise program only (EX).\n\nAge (years), tall (cm), weight (kg), and BMI (kg/m2), BMD of the lumbar spine (L2-L4) and entire hip, HRQoL, and fall risk, were all measured in all participants. Bone tests such s-OC, s-NTX, s-CTX, BSAP, PTH, and Vit D were assessed Pre-treatment, 12-weeks post-treatment, and six-months as follow-up estimations were taken from all groups.\n\nBMD was measured in all applicants at the lumbar spine (L2-L4) and overall hip pre-treatment, after 12 weeks of therapy, and at six-months as a follow-up utilizing a PRIMUS DEXA densitometer (OsteoSys, CO. LTD Korea). The manufacturer's Middle East (ethnicity) reference database was applied to determine T-scores for assessing osteopenia and OP. Before the assessments, the machines were calibrated on a daily basis applying spine phantoms provided by the company. Throughout the trial, the same operator performed all measures on all participants.\n\nBlood samples were collected from all applicants by means of plan vacationer tubes and handled to harvest serum, which was put away at −20°C until required for examination of osteocalcin (s-OC), N-terminal telopeptide of type I collagen (s-NTX), carboxy-terminal collagen crosslinks (s-CTX), bone-explicit antacid phosphatase (BSAP), parathyroid hormone (PTH), and 25-hydroxy nutrient D (Vit D). All examples were dissected in three-time.\n\nA self-controlled ECOS-16 survey was used to assess all patients' HRQoL. ECOS-16 has been shown to have high psychometric qualities and is a helpful tool for assessing HRQoL in older people with OP in both research and clinical practice. Physical function, pain, anxiety of sickness, and psychosocial work are all addressed in the ECOS-16 questionnaire. Each item's score runs from 1 (best HRQoL) to 5 (worst HRQoL).\n\nA Biodex balance device was used to assess the risk of falling (Biodex, New York, USA). A fall hazard test protocol is an accessible test that measures the stability of participants over 50 with varying balancing abilities and allows for the detection of potential falls. While the platform was moving, the applicants stood on the stand and concentrated to keep their base of gravity inside the middle of the bottom of support. The stage stability is rated from 1 to 12, with 1 being the most unstable. The platform becomes less stable as the resistance level drops. The test lasted 20 seconds; the stage was eight, and the attitude was bilateral with eyes open. The applicant did three test replications with a 30 second break between sets. The results were assessed to those of an age-corresponding healthy person.\n\nFull-body pulsed electromagnetic field\n\nA 68″×23″×1.5″ mat applicator and an 8″×10″×2.5″ pillow applicator from Sedona Pro PEMF Systems, Portland Oregon USA Ltd, were used to apply PEMF to the full body. The complete-body full strength is 30 gausses (3,000 micro-Tesla), while the pillow is 101 gausses (10,000 micro-Tesla). This mat emits a PEMF with a frequency range of 0.01-15,000 Hz, using sinusoidal, rectangular, multi-resonance, impulse, or sawtooth waveforms (10,000 micro-Tesla). Each participant lay on the mat for 30 minutes three points a week for a total of 12 weeks.19\n\nHigh-intensity laser therapy\n\nHILT was delivered by a HIRO 3 (Asalaser, Arcugnano, Italy). A pulsed Nd-YAG laser with a highest power of 3 kW, an usual power of 10.5 W, a wavelength of 1064 nm, a fluency level of 510-1780 mJ/cm2, a brief duration of 120-150 ls, a low frequency of 10-30 Hz, 0.1 percent duty cycle, a probe diameter of 0.5 cm, and a spot size of 0.2 cm2 are all included in the HILT machine. For a time of 12 weeks, all applicants received three sessions per week. The scan was done longitudinally and transversely in the lower back region to encompass the lumbar vertebrae, paraspinal muscles, and upper part of the gluteus maximus. The anterolateral, lateral, and posterolateral parts of the proximal hip region were also treated with the laser. A total dose of energy of 3000 J was delivered in two treatment stages. The primary period was completed with rapid manual imaging at 510, 610, and 710 mJ/cm2 in three successive subphases and 500 J in each subphase for a total of 1500 J. The last stage was similar the primary stage excluding that the imaging was slow. The mean area for the higher thigh or lower back was 200 cm2 with a normal fluency of 15 J/cm2, and the treatment time for each region was about 18 minutes. HILT was calibrated for constant output throughout the study period by the manufacturer.3\n\nExercise training program\n\nThe exercise schedule lasted 12 weeks and comprised of three 60-minute meetings per week, all of which took place in the exercise lab under the supervision of the researchers. Each session started with a 40-minutes including (flexibility, aerobic, strengthening, weight-bearing, and stability exercises), followed by a WBV training exercise. Starting with a warm-up and progressively increasing the strength of the training session is recommended in the initial training program. Patients in all groups were trained to do the similar exercise schedule at home once per day. WBV training involved a low-rate (30-40 Hz) vibration incentive delivered in a controlled setting (2-4 mm peak to peak). The pulsation spotlight began with one set of 30 seconds at 2 mm amplitude and 30 Hz, then progressed to two sessions of 5 minutes each at a high amplitude of 35 Hz. At the end of the session, they engaged in a cool-down phase that included relaxation and stretch exercises. All of the patients were encouraged to walk for half an hour every day. They were given a booklet describing the exercises, as well as a report on exercise compliance. Patients would be dropped out of the study if they missed three consecutive exercise sessions. Refer to the previous study for a more detailed description of the exercise protocol.19\n\nBased on the power study, G-Power 3.1 for Windows was applied to determine the expected sample size. SPSS for Windows, version 21 (statistical Package of Social Science, IBM, USA), was applied to analyze patient demographic information such as age, weight, height, and BMI. To compare intervals between management groups, an ANOVA with a post hoc Bonferroni test was implemented. The starting point, after-therapy, and six-month follow-up evaluations in each group were calculated applying a frequent measures ANOVA with a post hoc Bonferroni test. The degree of significance was approved at 0.05 for all groups.\n\n\nResults\n\nFor this study,59 a total of 110 men were selected as potential participants (Figure 1). The presence of 15 were ruled out (nine not meeting the inclusion criteria, and five refused to participate). A total of 95 men were randomly allocated to one of three groups in this study. Between the three groups, there were no significant differences in mean age, weight, height, BMI, or vitamin D levels (Table 1). After treatment, all participants were 100% adherent with their exercise programs.\n\n Non-significant differences in the same measurement interval among treatment groups (one-way ANOVA; P > 0.05).\n\nPretreatment scores for BMD of the lumbar spine and total hip between the three groups were not significantly different (P>0.05) at baseline (Table 2). There were significant differences (P<0.05) when comparing pre and post scores in the PEMF+Ex and HILT+EX groups after 12 weeks and six months of treatment, with significant improvement in the PEMF +EX group over the HILT+EX group, but no significant differences (P>0.05) in the EX-group (Table 2).\n\n Non-significant differences.\n\nAt baseline, there were no significant differences in HRQoL scores between the three groups (P>0.05) (Table 2). There were significant differences (P<0.05) in the three groups' baseline, 12 week, and six-month scores, with significant improvement in the PEMF+Ex group compared to the HILT+EX and EX groups, as shown in Table 2.\n\nAs shown in Table 2, there were no significant differences (P>0.05) in pretreatment fall risk scores between the three groups at baseline, but there were significant differences (P<0.05) in the three groups after 12 weeks of treatment and after six months of follow up after treatment, with significant improvement in the PEMF+Ex group compared to the HILT+EX and EX groups.\n\nThe s-OC, s-NTX, BSAP, PTH were declined markedly in the PEMF+ EX group after treatment in comparison with baseline values (P<0.001). While the total calcium and 25(OH) VD were non-significant difference at baseline, and 12 weeks after treatment in the PEMF+ EX group (P>0.05). Regarding the HILT+EX group the s-OC, s-NTX, BSAP, PTH were significantly reduced after treatment in comparison with baseline values (P<0.001). Meanwhile the total calcium and 25(OH) VD were insignificantly change at baseline, and 12 weeks after treatment in the HILT+EX group (P>0.05). Simultaneously the amount of s-OC, s-NTX, BSAP, PTH showed significant reduction in EX group after treatment in compare with baseline values (P<0.001). On the other hand, total calcium and 25(OH) VD reveled non-significant difference at baseline, and 12 weeks after treatment in the EX-group (P>0.05) as displayed in Table 3.\n\n** Non-significant differences.\n\nIn the post-treatment group, the s-OC, s-NTX, BSAP, PTH were significantly reduced in the PEMF+ EX when compared with the HILT+EX and EX groups. Also, those parameters were significantly reduced in the HILT+EX as compared with EX group. While the total serum calcium and 25(OH) VD were non-significant change between all treated groups as showed in Table 3.\n\nThe S-OC was significant decreased in the PEMF+ EX group as compared with HILT+EX and EX groups after follow-up at six months. The s-NTX was reduced markedly in the PEMF+ EX and HILT+EX when compared with EX. While the BSAP and PTH were significantly lowered in the PEMF+ EX groups compared with HILT+EX and EX groups after follow-up six months, while those parameters were lower in the HILT+EX than the EX-group. The total calcium and 25(OH) VD were non-significant change between all groups after six months follow-up as displayed in Table 3.\n\n\nDiscussion\n\nThe study's main findings show that following 12 weeks of treatment, PEMF combined with exercise has a greater effect than HILT+EX or EX alone in boosting BMD and enhancing bone formation, suppressing bone-resorption indicators, improving quality of life, and reducing fall risk, with these effects persisting up to six months after treatment.\n\nThe effects of PEMF on living organisms are well documented. Cells in organisms have membrane that electrically charged and closely controls the electrically charged ions concentration which act as potent signal mediators, such as Ca+2 or Na+.25 As a result, it's believed that the membrane level is site of the majority of PEMF effects in cells. PEMF has been proven to act on the concentrations and dependent pathways of Ca+2,26,27 while Varani et al. have recently demonstrated that PEMF regulates Adenosine receptors.28 In fact, the recent evidence that the transducing PEMF effects in cells is the role for primary cilia by Yan et al.29 and Xie et al.30 could be one of a wide activity on membrane passaging, which includes receptor trafficking. PEMF has been demonstrated to change defenses against oxygen species reaction,31 bioactive factor synthesis, and pathways between cells such as the sAC–cAMP–PKA–CREB signaling pathway.21\n\nPEMF stimulation enhances the anabolic actions in skeleton of the unloaded hindlimb of rats, as proved by biochemical, mechanical, micro-computed tomography, and morphometric results, according to Jing and colleagues.32 They further show that portmanteau signaling may be involved in PEMF's anabolic actions on bone. PEMF, with no side effects method, could become a useful treatment modality for osteopenia and/or osteoporosis, as well as a countermeasure for bone loss, according to their findings.32\n\nRecently in a review, the PEMFs signaling pathways in bone repair have been summarized, including Ca+2, Wnt/-catenin, mitogen-activated protein kinase, fibroblast, vascular endothelial and transforming growth factor/bone morphogenetic proteins, insulin-like growth factor, and cAMP/protein. Also, the mammalian target of the pathway of rapamycin has been shown to be the PEMF signaling pathway involved in bone building. PEMF exposure affected the formation of growth factors, enhancing the formation of extra-cellular matrix proteins and enhancing tissue repair.15\n\nPhysical therapy has gained more attention in recent years, in addition to pharmacotherapy, for this type of disease.33 PEMF has been shown to have potential effect in the treatment of OP21,34 as an effective physiotherapy for various disorders of bone. Management of postmenopausal osteoporosis with PEMF at particular dosage (8 Hz, 3.82 m T, 40 minutes per day, and six sessions per week) had the same treatment effect for 24 weeks, according to a randomized, active-controlled clinical trial.34\n\nEbid et al. concluded in a recent randomized controlled study of osteoporotic men that PEMF with an exercise program has an obvious role in management of osteoporosis, in increasing BMD, and enhancing bone formation than exercise alone and PEMF alone, and suppresses the markers of bone-resorption following 12-weeks of treatment, and the effect continued up to six months.19\n\nBecause of its efficacy and non-side effects, PEMF has a lot of potential as alternative treatment method for musculoskeletal problems. Many studies have investigated into the PEMF stimulation on various musculoskeletal disorders including underlying cellular and subcellular mechanisms, as a molecular basis presenting its clinical application. The unknown issues as the deeper mechanisms and the optimal using parameter, remains unresolved. As a result, further research from well-designed, high-quality studies is necessary before they can be widely used in clinical situations to know the deeper mechanisms, the best treatment dosage, and establish the ideal protocol for decision-making about health-care. In conclusion, with suitable patient selection, well defined indications, and consistent treatment strategies, PEMF have the potential to play a significant role in the management of musculoskeletal disorders in the future.18\n\nComparing to baseline scores, the combined HILT and exercise effectively improve the lumbar and hip BMD. By stimulating cytochrome oxidase in the mitochondrial activity of osteoblast cells,35 laser treatment can modify cellular metabolism by contributing in the production of an electrochemical potential and the production of oxygen molecules that are utilized to generate adenosine triphosphate.36 Lasers stimulate proliferation by providing energy to tissues and growth factor secretion, as well as improve tissue function by enhancing DNA and RNA synthesis,37 resulting in new bone formation and enhanced osteopenic fracture repair.35\n\nPrevious HILT studies have shown this to be effective for treating musculoskeletal pain with no adverse effects or histological risks. The unique properties of HILT enable a laser with a wavelength of 1064 nm and pulsed high-power to penetrate deeper layers, resulting in laser energy diffusion in all directions, stimulating cytochrome oxidase and increasing mitochondrial oxidative reaction while enhancing photochemical and photobiological effects.38,39 Furthermore, with low-frequency high-power emissions, very brief durations, and paused time intervals, HILT are believed to have a photomechanical effect and repetitive mechanical stimulation may can reach the bone tissue.40\n\nThe mechanoreceptive cells in bone tissue, known as osteocytes, adapt to mechanical stimuli by transmitting signals through the bone matrix to stimulate osteoblast bone formation. This mechanical stimulation may help to promote bone health by increasing or maintaining BMD.41 The study's results are in agreement with previous studies, that found HILT in combination with exercise was better than exercise alone in enhancing BMD of lumber region following treatment for 24 weeks and its effects continue up to a year.42\n\nIn comparison to baseline scores, the combination of HILT and exercise successfully reduces the risk of fall and improves quality of life post-treatment. HILT may have the impact of reducing exercise-related pain as well as relieving accompanying issues such as muscular spasms and back pain.43 The calming effect of HILT may be due to its ability to reduce pain impulses conduction and increase the rate of substance production in tissue, which is comparable to morphine's action.17 It may also improve circulation, the permeability of blood vessels, and cellular metabolism by blocking pain transmission through Ad- and C-fibers.44\n\nHILT may improve muscular efficiency during exercise and help muscle recovery from exercise-induced fatigue.43 Regular physical activity enhanced strength, balance, and proprioception.45 Exercise regimens may also help people reduce their risk of fall.46 Programs of moderate intensity exercise designed specifically for preventing falls improved balance, the strength of the muscles, ambulation, and decrease fall risks, according to previous studies.47\n\nThe findings of this study are consistent with previous studies that found that exercises improved BMD and decrease fall risk in women with OP.15 In addition, the current study found that the group of HILT had a better quality of life in comparing with the exercises group, as demonstrated by higher scores in all five EHP-5 domains and a significant difference in the quality-of-life scores mean value, which is consistent with previous research.48\n\nIn comparison to pre-treatment scores, exercise effectively increased both lumbar and total hip BMD in men with OP following treatment of 12 weeks and at six-months later, but there was no significant effect between 12 weeks and six-month follow-up scores in this study. The results of this study, which were in agree with the previous clinical trials, suggest that exercise had a small, statistically significant, but potentially obvious effect on postmenopausal women with OP.49\n\nA significant and relevant long-term effect on bone density at the lumbar region and neck of femur was demonstrated with an exercise program adapted to the subject's priorities.50 Although there is no linear effect, an increase in daily living activity using simple, performed activities can help prevent decreases of post-menopausal BMD.51 Mechanical loading exercise regulates bone vascularization by regulating angiogenic mediators, which are important for skeletal health.52 Exercise enhanced BMD in the neck of femur and lumbar region by 1.8 % and 1.5 %, respectively, with a significant increase in muscle strength.53 Resistance training combined with weight-bearing activities may help prevent osteoporosis by preventing or delaying the loss of BMD in men at middle-aged and older.54\n\nExercises also enhance muscle strength, as well as static and dynamic balance. The findings of this study opposed previous clinical trials that suggested resistance exercises does not improve femur bone density more than walking for 30 minutes three times a week.55 Differences in age ranges, as well as the kind, period and intensity of exercise, may explain the differences between results of this study and those of previous studies.\n\nIn comparison to baseline scores, the exercise effectively reduces the risk of fall and improves quality of life. Resistance training decrease pain and enhance wellbeing, mood status, and cognitive abilities, improve quality of life,56 and lower falls risk,57 all of which reduce the risk of fracture,58 according to the results of this study.\n\n\nConclusions\n\nPEMF in combination with program of exercise is better than HILT combined with exercise or exercise alone in improving BMD and promoting bone synthesis, suppressing bone-resorption markers, and improving quality of life and risk of fall following treatment of 12-weeks and the effect continue up to six months.\n\nThe benefits of such treatments should be studied for extended periods of time in various musculoskeletal disorders, as well as in women with osteoporosis. As a result, further research from well-designed, high-quality studies is needed to understand deeper mechanisms, standardize treatment parameters, and determine the appropriate techniques for health-care decision-making before they can be widely used in healthcare situations.\n\nIn terms of limitations, the findings of this investigation should be regarded with caution because our trial's six-month follow-up time was insufficient to determine the efficacy of PEMF and HILT on BMD and bone turnover indicators. Bone metabolism takes a long time to adapt to exercise, according to previous research. All of the study participants were instructed to maintain a well-balanced diet and follow a home exercise program that included 30 minutes of daily walking and exercise compliance monitoring. Because none of the participants reported any drawbacks, major side effects, or new-onset local discomfort, we regarded the home-based exercise program to be a limiting factor in this study. Although the results of this study are encouraging, more trials with fewer inclusion and exclusion criteria would yield more generalizable results. Long-term research as well as comparisons to different therapies are warranted.\n\n\nData availability\n\nFigshare: Age, height and weight raw data, https://doi.org/10.6084/m9.figshare.16920130.59\n\nFigshare: BMD total hip raw data, https://doi.org/10.6084/m9.figshare.16920151.\n\nFigshare: BMD lumbar spine raw data, https://doi.org/10.6084/m9.figshare.16920163.\n\nFigshare: Bone markers raw data, https://doi.org/10.6084/m9.figshare.16920184.\n\nFigshare: HRQoL raw data, https://doi.org/10.6084/m9.figshare.16920190.\n\nFigshare: Fall risk raw data, https://doi.org/10.6084/m9.figshare.16920196.\n\nFigshare: Tables, https://doi.org/10.6084/m9.figshare.16920250.\n\nFigshare: CONSORT Checklist, https://doi.org/10.6084/m9.figshare.16920253.\n\nFigshare: Flow chart of the study, https://doi.org/10.6084/m9.figshare.16920232.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThis work was funded by grant number 15-MED5406-10 from the National Science, Technology and Innovation Plan (MAARIFAH), the King Abdul-Aziz City for Science and Technology (KACST), Kingdom of Saudi Arabia. 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Eng. 2011; 71.\n\nThabet AAE, Alshehri MA: Effect of Pulsed High-Intensity Laser Therapy on Pain, Adhesions, and Quality of Life in Women Having Endometriosis: A Randomized Controlled Trial. Photomed. Laser Surg. 2018 Jul; 36(7): 363–369. PubMed Abstract \| Publisher Full Text\n\nBenedetti MG, Furlini G, Zati A, et al.: The Effectiveness of Physical Exercise on Bone Density in Osteoporotic Patients. Biomed. Res. Int. 2018; 2018: 1–10. Publisher Full Text\n\nKemmler W, Engelke K, von Stengel S : Long-Term Exercise and Bone Mineral Density Changes in Postmenopausal Women--Are There Periods of Reduced Effectiveness?. J. Bone Miner. Res. 2016; 31(1): 215–222. PubMed Abstract \| Publisher Full Text\n\nMuir JM, Ye C, Bhandari M, et al.: The effect of regular physical activity on bone mineral density in post-menopausal women aged 75 and over: a retrospective analysis from the Canadian multicentre osteoporosis study. BMC Musculoskelet. Disord. 2013; 14: 253. 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Publisher Full Text" }	[ { "id": "120908", "date": "07 Feb 2024", "name": "Hayam Mahmoud Sayed", "expertise": [ "Reviewer Expertise physical therapy for Neurology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an original research study on a significant topic. The research goal is applicable. The materials and methods are well described, and the data interpretation is proper. The study's goal is addressed in the conclusion, I appreciate this an excellent paper, but I have a few reservations.\nThere is no need for more information about participant characteristics in the abstract, such as (mean age, 52 years; mean height, 176 cm; mean weight, 83 kg; mean body mass index (BMI), 26.86 kg/m2), as stated in the method section of the research.\n\nWhat do you mean by a change in their pharmacological treatment in the previous three months in the method section (participants section)? Is the participant taking any medications that have an effect on bone density during the study?\n\nWhat is the complete name of the ECOS-16 survey? It is preferable to define it.\n\nDoes the difference in treatment session time between electromagnetic and laser affect the outcome on the electromagnetic side?\n\nIs there a period of rest between different types of exercises within the program?\n\nOsteoporosis and the fractures that accompany it are a major public health concern around the world. OP is estimated to affect over 200 million people worldwide, resulting in over 8.9 million fractures each year.4 (this ratio belonging to KSA not for world).\n\nReduce their alcohol consumption.12 (Saudi males are not used to drinking alcohol, please delete this recommendation).\n\nIf applicants had physical treatment or a shift in their pharmacological treatment in the prior three months, they would not be allowed to use any extra treatments, specific regimens, or aerobic training options for the duration of the study. (How could you give them exercises as you do not allow them for any extra exercises) - would you give more explanation?\n\nA self-controlled ECOS-16 survey was used to assess all patients' HRQoL. ECOS-16 has been shown to have high psychometric qualities and is a helpful tool for assessing HRQoL in older people with OP in both research and clinical practice. (would you give a Reference please?)\n\nThe results were assessed to those of an age-corresponding healthy person. (Explain).\n\nThe pulsation spotlight began with one set of 30 seconds at 2 mm amplitude and 30 Hz, then progressed to two sessions of 5 minutes each at a high amplitude of 35 Hz. At the end of the session, (would you give more clarification about actual time and pattern of vibration?)\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-86
https://f1000research.com/articles/11-83/v1	24 Jan 22	{ "type": "Research Article", "title": "Parametric modelling of rainfall return periods in south-western Nigeria: Survival analysis approach", "authors": [ "Phillip Awodutire", "Blessing Sasanya", "Olohita Ufuoma", "Oluwafemi Samson Balogun", "Blessing Sasanya", "Olohita Ufuoma", "Oluwafemi Samson Balogun" ], "abstract": "Background: Rainfall is the main source of water on the earth’s surface. It infiltrates and percolates deep into the soil for groundwater recharge. Rainfall patterns, amounts, durations, and intensities can vary daily, monthly, annually, and spatially. It is therefore important to accurately estimate rainfall return periods, which can be employed in hydraulic design and flood control measures. Methods: This research considered the survival analysis approach for the prediction of rainfall return periods including intensity, and months during which these would occur in south-western Nigeria. Twenty years’ of annual rainfall data were obtained from three metrological stations and these were subjected to nine different probability plotting position methods. Results from the plotting positions was further subjected to four survival models using five years of censor time. The Akaike Information Criterion (AIC) was used to determine the best-fitting model for the dataset. Results: The Laplace probability plotting position in conjunction with the log-logistic distribution best describes the datasets, since it gave the lowest AIC value of 22.53. The log-logistic distribution is also suitable for the prediction of return period from the Weibull probability plotting position since the AIC values were 6.934 and -4.332 respectively. The Hirsh plotting position in conjunction with the Weibull distribution is also suitable for the description of the dataset. Conclusion: The established parametric models are suitable for the accurate prediction of return periods of peak rainfall events during any month of the year.", "keywords": [ "return periods", "peak rainfall intensities", "survival analysis", "parametric modelling", "probability plotting position" ], "content": "Introduction\n\nThe concept of survival analysis as a statistical tool in handling time to an event related problem was initially designed for medical related studies. Example of such studies include time taken for a patient to recover from diseases, time to die from a disease, and the likes. Several studies have been conducted using parametric models to analyse health related issues (Awodutire et al., 2018; Naseri, et al., 2018). In recent times, survival analysis has been extended to finances (Laitinen, 2005; Witzany et al., 2012; Lee, 2014) engineering (reliability analysis, Awodutire et al., 2021), politics (Jonathan, 2014) and similar. It may be of interest to model the relationship of time to event to other covariates (sometimes called predictors). For this research, the event of interest was rainfall intensities and its return period (i.e. the time taken to experience rainfall).\n\nNigeria is a country with many rivers, lakes, ponds (natural and manmade) and tides of these rivers are controlled majorly by seasons. There are different climatic seasons in Nigeria namely dry and rainy seasons. During the rainy seasons, rainfall amounts, and intensities are usually very high. Nigeria’s economy is linked to climate sensitive activities (Houessou-Doussou et al., 2019). Precipitation (rainfall) is formed when saturated air is heated, rises by a mountain, convectional current or frontal action (Ogungbenro and Morakinyo, 2014). Rainfall is the main source of water on the earth’s surface which percolates through the soil for groundwater recharge and runs off the soil surface forming surface water such as streams and rivers (Salase et al., 2015). It also forms depression storages, it is trapped by vegetative interception and it is then transferred from the land to the atmosphere by evapotranspiration. The importance of rainfall in water resources management, water supply, agricultural activities and food production cannot be over emphasized; however, rainfall varies spatially and temporally. Precipitation is the main driver of variability in the water balance over time and space (Davie, 2008).\n\nRainfall can be of immense benefit to the environment, but it can also be accompanied by adverse effects at certain times. Excessive rainfall can lead to undesirable disasters such as flooding and landslides. Rainfall patterns, amount, duration and intensity can vary daily, monthly, annually and from one place to another. This makes it subject to uncertainties and probabilities, which are partly explained by the concept of rainfall return periods. Return period is an essential tool in hydrology which estimates the time interval between events of similar sizes or intensities (Laura and Richard, 2015). An understanding of historical and current rainfall trends is essential in determining the return periods in a particular area. Factors such as missing data and unknown probability distribution function of annual peaks makes the estimation of return period of real events a tedious task; frequency analysis is thus used to estimate the return periods of specific events (Houessou-Doussou et al., 2019). Consequently, it is important to estimate rainfall return period by modelling its components with real world behaviours and attributes. Varying precipitation can influence hydrology, water resources as well as extreme events such as flood and drought. Statistical estimates are therefore used for forecasting, prediction, correlation, collation and analysis of daily, monthly or annual rainfall rates and duration data (Ybanez, 2013). Analysis of past rainfall data provides estimates of recurrence interval, which can also be used to predict into the future (Olatunde and Adejoh, 2017). According to Olatunde and Adejoh (2017), stochastic analysis of rainfall is of high importance in the design and development of civil engineering structures such as buildings, bridges, water storage structures (reservoirs, detention basins, rainwater tanks). These structures are needed to maintain continual usage, under specified reliability, environmental or agricultural conditions. Probability analysis of past rainfall records are useful in that regard for the determination and prediction of highest rainfall months and years (Ewemoje and Ewemoje, 2011). These are also important for preparation against disaster.\n\nGenerally, stochastic analysis involves characterization of the probability distribution of the variable (rainfall in this case) and its associated predictors, so that conditional probability distribution can be derived. For rainfall, the characterization is more specific to the time scale being modelled, which could be annual, monthly, daily or sub daily time scales (Olatunde and Adejoh, 2017).\n\nSeveral studies have been done around rainfall prediction from past records using probability functions. Ewemoje and Ewemoje (2011) researched the best plotting position and distribution for flood estimation of the Ona river using 18 years’ peak flood data from the Ogun Oshun River Basin, Nigeria. Three probability distributions and plotting position methods were compared. The suitability of the Hazen, Weibull and California probability position methods were compared, as well as those of the normal, log-normal and log Pearson type (III) distributions. The Hazen plotting position and log Pearson type (III) distribution performed best. Hurford et al. (2012) studied the validation of return period of rainfall thresholds used for extreme rainfall alerts using links with rainfall intensities and observed surface flood events. The research hinged on investigating if return period is adequate for the warning of surface water flooding by examining the intensity and return period of rainfall associated with observed surface water flood events. Rainfall amount recorded by rain gauges and flood events were analysed which showed that most surface flood water events were associated with rainfall intensities of less than a 1-in-10-year return period. It was concluded that better understanding of the relationship between flood magnitude and rainfall intensity could be enhanced through the improvement of data recording on flood magnitude and duration for informed comparison between surface water flood warning thresholds. Agbede and Aiyelokun (2016) established the most suitable stochastic model for flood management in the Yewa sub-basin, south-western Nigeria. The peak floods were fitted into normal, gamma, gumbel and Weibull distributions using 13 years’ peak flood data, with return periods obtained from the Hazen plotting position method. The Weibull distribution was reported to be the most suitable distribution for predictions of flood in the Yewa sub-basin. In the same vein, Aiyelokun et al. (2017) fitted 31 years’ hydrologic data from the gauged Opeki river to various probability distributions using return periods founded by the Hazen method. The researchers employed normal, log normal, log Pearson type (III), exponential, extreme value type (I), extreme value type (II) and the three-parameter burr distribution. The exponential and normal distributions were reported incapable of predicting flood flows from the Opeki river. It was further reported that the log Pearson type (III) distribution was the most suitable for the estimation of peak flood from the Opeki river. Santos et al. (2015) analysed seasonal return periods for maximum daily precipitation in the Brazilian Amazon. The extreme value theory was adopted using the non-parametric generalized extreme value (GEV) distribution and the generalized Pareto distribution (GPD). The GEV and GPD goodness of fit were evaluated by applying the Kolmogorov–Smirnov (KS) test, which compares the cumulative empirical distributions with theoretical ones. The KS test indicated that the tested distributions had a good fit, particularly the GEV distribution. They were thus adequate for the study of seasonal maximum daily precipitation. Furthermore, Yahaya (2012) and Ogungbenro and Morakinyo (2014) used statistical methods to justify the changes observed in monthly and annual rainfall trends over some years. Obot (2010) used the non-parametric Mann-Kendall test to check for significant trends in rainfall in Nigeria in some randomly selected locations.\n\nHaving reviewed these studies, none used the survival analysis approach to determine possible return period of maximum or peak rainfall events. Several studies have predicted return periods of rainfall from complete datasets, but according to Houessou-Doussou (2019), there may be missing or censored data. Therefore, this study aims to develop models which can adequately predict return periods from peak rainfall intensities and the months of occurrence of such peaks in south-western Nigeria in cases of missing or censored information. The return periods were achieved by plotting probability positions for obtained rainfall data using nine different methods. The plotting position methods were compared and subjected to statistical analysis using parametric modelling which compared four survival models. The parametric approach has been proven over time to be the best method of analysing time data events. This has resulted from its ability to handle datasets with minimal sample sizes and its efficient and consistent estimations. Studies applying survival analysis to rainfall data are rare, but this study takes this approach for the analysis of this time data event.\n\n\nMethods\n\nThe return periods of annual peak rainfall intensities were studied. Monthly rainfall data were obtained from three meteorological stations in south-western Nigeria. The peak intensities from 2009–2018 were considered and their corresponding months of occurrence recorded (Awodutire et al., 2021a). The annual maximums of the monthly rainfall intensities were arranged in descending order of magnitude. These were subjected to probability plotting positions by comparing the California, Weibull, Hazen, Adamowski, Blom, Chegodagev, Gringoten, Hirsh, and Laplace methods. These are given by equations (1) to (9).\n\nWhere m = rank order of the rainfall intensities, n = number of years of record, T = return period (years). For this research, the return period is censored at five years. The parametric survival model (also known as accelerated failure time model (AFT) is of the form:\n\nFor this study, four different parametric survival models were employed for both return periods generated from the probability plotting equations. These are Exponential, Weibull, log-normal and log-logistic parametric models described by the equations 11 to 14.\n\nThe Akaike Information Criterion (AIC) was used for the comparative studies of the resulting models. The AIC is given as equation 15:\n\nWhere L is the likelihood value of the model.\n\nThe model with the lowest AIC performed the best. The significance of the independent variables in the model (contribution to the dependent variable) were assessed at 0.05 significance level with hypothesis as H0: ρi = 0 vs H0: ρi ≠ 0. The H0 was rejected at p<0.05. Data analysis was conducted using SPSS version 20.0 (IBM Corp. Released, 2020) (RRID:SCR_019096) and R 4.10 programming (R core team, 2017) (RRID:SCR_001905). The R code for data analysis is presented in Awodutire et al. (2021b).\n\n\nResults and discussion\n\nThe average highest rainfall intensity from the data obtained from the weather stations was 453.1 mm/month experienced in August 2015. while the least rainfall intensity was 201.1 mm/month in July 2017. The return periods of the highest rainfall intensity were 20, 21, 40, 27.33, 32.4, 29.14, 35.92, 16.8 and 11 years respectively for the California, Weibull, Hazen, Adamowski, Blom, Chegodayev, Gringorten, Hirsh and Laplace probability plotting position methods. The return periods of the lowest rainfall intensity were 1.00, 1.00, 1.03, 1.03, 1.03, 1.03, 1.03, 1.04 and 1.05 respectively for the California, Weibull, Hazen, Adamowski, Blom, Chegodayev, Gringorten, Hirsh and Laplace probability plotting position methods. Tables 1–9 show the various parametric models relating rainfall return period, monthly intensities and month of the year during which the peak rainfall intensity was experienced. Parametric models capable of adequately predicting rainfall return periods from rainfall intensities and annual calendar months were obtained from the log-normal, exponential, log-logistic and Weibull distributions. They are of the form shown on equation 12:\n\nT is the return period, C is the intercept of the equation, B is the coefficient of x1, which is the rainfall intensity, A is the coefficient of the month variable x2.\n\nParametric models were derived by comparing four probability distributions using return periods from the nine probability plotting position methods. Right censoring was considered. The rainfall return period was censored at five years. The p-values in Tables 1–9 for each survival model under consideration indicate that the models fit the data very well (Awodutire et al., 2021c). The AIC values were used to compare the models from each of the distributions and the probability plotting position methods.\n\nTable 1 compares the probability distributions employed for the derivation of the suitable parametric model from the California probability plotting positions. The log-logistic distribution proved to be the most suitable distribution for the plotting position since it has the lowest AIC value of 22.53. In the same vein, the log-logistic distribution is most suitable for the prediction of return period from the Weibull and Laplace probability plotting position methods since the AIC values were 6.934 and -4.332 respectively. The exponential distribution however had the highest AIC values for both the California and Weibull plotting position methods. The AIC values were 62.56 and 61.40 respectively. The exponential distributions proved to be most unsuitable for all the plotting position methods. The AIC values of the exponential distribution consistently ranged between 61 and 65. The Weibull distribution, however, was the best fit to the return period data from the Hazen, Adamoski, Blom, Chevgodayev, Gringoten, and Hirsh probability plotting positions. The AIC values attributed to the Weibull distribution were 16.18, 9.19, 12.16, 10.20, 14.14 and -0.23 for the respective plotting position methods. From the aforementioned results, it was inferred that the distributions which best fitted the models were the log-logistic and the Weibull probability distributions. The Laplace probability plotting position in conjunction with the log-logistic distribution best described the datasets. This is in conformity with the model equation derived by Hurford et al. (2012). The Hirsh probability plotting position in conjunction with the Weibull probability distribution was also suitable for the description of the dataset and prediction of return period from the rainfall intensities and month of occurrence of such intensities. This is described by the model equation 18). It must, however, be noted that the censor period for this study is five years. This means that any rainfall return period predicted from the equations exceeding five years must be censored according to the theory of survival analysis.\n\nThese findings are slightly contrary to the report of Obot (2010), which reported that the Weibull probability position in conjunction with the normal distribution gave the highest fit for the Apoje sub-basin of Osun River. The combinations were reported to result in an R2 value of 0.9950 and root mean square error (RMSE) value of 35.09 m3/s. This is contrary to the findings of Agbede and Aiyelokun (2016) who compared the Hazen, Weibull and California probability plotting positions using the normal, log-normal and log Pearson type III distribution for the prediction of flows in Ona river. The Hazen plotting position method was reported to perform best since it gave a higher regression coefficient (R2) and minimal RMSE value. The log-Pearson (III) distribution gave the least absolute difference for all the plotting positions compared for the study. Adeboye and Alatise (2007) compared seventeen probability plotting positions using the Gumbel distribution. The study reported that the Hazen plotting position was the best for sample sizes ranging from 10 to 20.\n\n\nConclusions\n\nThe results obtained from the analysis of rainfall intensity and return period revealed that parametric models are essential tools for the estimation of time intervals between extreme and peak rainfall events in different months of the year. The combination of the Laplace plotting position and log-logistic distribution or the Hirsh plotting position and Weibull distribution fitted the datasets best. The established parametric models were suitable for the accurate prediction of return periods of peak rainfall events during any month of the year. The accelerated failure time approach was found to be suitable for the analysis of rainfall data by determining the best of several parametric models. In this research, the parametric models employed showed the relationships between rainfall return periods, rainfall intensity and month of the year.\n\n\nData availability\n\nZenodo: Underlying data for ‘Parametric modelling of rainfall return periods in south-western Nigeria: Survival analysis approach’.\n\nThis project contains the following underlying data:\n\n• Data file: rain.csv https://doi.org/10.5281/zenodo.5797868 (Awodutire et al., 2021a)\n\n• Table 1: Parametric models of return period California probability plotting positions.\n\n• Table 2: Parametric models of return period Weibull probability plotting positions.\n\n• Table 3: Parametric models of return period Hazen probability plotting positions.\n\n• Table 4: Parametric models of return period Adamowski probability plotting positions.\n\n• Table 5: Parametric models of return period Blom probability plotting positions.\n\n• Table 6: Parametric models of return period Chegodayev probability plotting positions.\n\n• Table 7: Parametric models of return period Gringorten probability plotting positions.\n\n• Table 8: Parametric models of return period Hirsh probability plotting positions.\n\n• Table 9: Parametric models of return period Laplace probability plotting positions.\n\nhttps://doi.org/10.5281/zenodo.5799930 (Awodutire et al., 2021c)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.5800018 (Awodutire et al., 2021b)\n\nLicense: Creative Commons Attribution 4.0 International", "appendix": "References\n\nAdeboye OB, Alatise MO: Performance of Probability Distributions and Plotting Positions in Estimating the Flood of River Osun at Apoje Sub-basin, Nigeria. Agricultural Engineering International: The CIGR E Journal. 2007; IX. July, 2007.\n\nAgbede OA, Aiyelokun OO: Establishment of a stochastic model for sustainable economic flood management in Yewa sub-basin, Southwest Nigeria. Civil Eng. Journal 2016; 2(12): 646–655. Publisher Full Text\n\nAiyelokun OO, Ojelabi A, Malomo S, et al.: Efficient flood forecasting for the operation of hydraulic structures in a typical river basin. Int. J. Sci. Eng. Res. 2017; 8(11).\n\nAwodutire PO, Olapade AK, Oladapo OAI, et al.: Assessing Survival Times of Breast Cancer Patients Using Type I Generalized Half Logistic Survival Model. JAMMR 2018; 25: 1–7. Publisher Full Text\n\nAwodutire PO, Balogun OS, Olapade AK, et al.: The modified beta transmuted family of distributions with application using exponential distribution. PLoS One 2021; 16(11): e0258512. Publisher Full Text\n\nAwodutire PO, Sasanya BF, Ufuoma OG, et al.: Dataset on Modelling Rainfall Return Periods and Intensity: Survival Analysis Approach [Data set]. Zenodo. 2021a. Publisher Full Text\n\nAwodutire PO, Sasanya BF, Ufuoma OG, et al.: Tables showing the results of the models using the different plotting positions. Zenodo. 2021b. Publisher Full Text\n\nAwodutire PO, Sasanya BF, Ufuoma OG, et al.: R Code on Modelling Rainfall Return Periods and Intensity: Survival Analysis Approach. Zenodo. 2021c. Publisher Full Text\n\nDavie T: Fundamentals of hydrology. Routledge Fundamental of Physical Geography. Ed. By John Gerrard. 2008.\n\nEwemoje TA, Ewemoje AS: Best distribution and probability positions for daily maximum flood estimation at Ona River in Ogun-Oshun River basin, Nigeria. Agric. Eng. Int. CIGR J. 2011; 2011: 3.\n\nHouessou-Doussou EAY, Gathenya JM, Njuguna M, et al.: Flood Frequency Analysis Participatory GIS and Rainfall Data For Two Stations in Narok Town, Kenya. Hydrology 2019; 6: 90. Publisher Full Text\n\nHurford AP, Parker DJP, Priest SJ, et al.: Validating the return period of rainfall thresholds used for extreme rainfall alerts by linking rainfall intensities with observed surface water flood events. J. Flood Risk Management 2012; 5(5): 134–142. Publisher Full Text\n\nIBM Corp. Released: IBM SPSS Statistics for Windows, Version 27.0 Armonk, NY: IBM Corp; 2020. Reference Source\n\nJonathan G: Survival Analysis and European Union Decision Making. European Union Poilitics. SAGE Publications; 2014; vol. 8. : 155–179.\n\nLaitinen EK: Survival Analysis and Financial Distress Prediction: Finnish Evidence Review of Accounting and Finance.2005. Publisher Full Text\n\nLaura KR, Richard MV: Reliability, return periods and risk under nonstationarity. Water Resour. Res. 2015; 51(8). Publisher Full Text\n\nLee M: Business Bankruptcy Prediction Based on Survival Analysis Approach. International Journal of Computer Science and Information Technology. 2014; 6: 103–119. Publisher Full Text\n\nNaseri P, Baghestani AR, Momenyan N, et al.: Application of a Mixture Cure Fraction Model Based on the Generalized Modified Weibull Distribution for Analyzing Survival of Patients with Breast Cancer. Int. J. Cancer Manag. 2018; 11: 2018. Publisher Full Text\n\nObot N, Chendo M, Udo S, et al.: Evaluation of rainfall trends in Nigeria for 30 years (1978-2007). Int. J. Phys. Sci. 2010; 5.\n\nOgungbenro SB, Morakinyo TE: Rainfall Distribution and Change Detection Across Climatic Zones in Nigeria. Weather Clim. Extremes. 2014; 5-6: 1–6. Publisher Full Text\n\nOlatunde AF, Adejoh I: Annual exceedance probability and return periods of rainstorms in Lokoja. Int. J. Soc. Sci. 2017; 11.\n\nR Core Team: R: A language and environment for statistical computing Vienna, Austria: R Foundation for Statistical Computing; 2017. Reference Source\n\nSalase AE, Agyimpomaa DEE, Selasi DD, et al.: Precipitation and rainfall types with their characteristic features. J. Nat. Sci. Res. 2015; 5(20).\n\nSantos EB, Lucio PS, Silva CMSE: Seasonal Analysis of Return Periods for Maximum Daily Precipitation in the Brazilian Amazon. J. Hydrometrol. 2015; 16: 973–984. Publisher Full Text\n\nWitzany Y, Rychnovsky M, Charamza P: Survival Analysis in LGD modelling. European Financial and Accounting Journal 2012; 7: 6–27. Publisher Full Text\n\nYbanez R: Understanding Rainfall Return Periods Project NOAH Open-File Reports 2013; 1: pp 3–4. 2362 7409.\n\nYahaya AS, Nor NM, Jali NRM, et al.: Determination of probability plotting position for type 1 extreme value distribution. J. Appl. Sci. 2012; 12(14): 1501–1506. Publisher Full Text" }	[ { "id": "120739", "date": "08 Feb 2022", "name": "Thomas Xavier", "expertise": [ "Reviewer Expertise Statistical distributions", "survival analysis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have used survival analysis for the prediction of rainfall return periods. Twenty years of annual rainfall data were obtained from three meteorological stations for the analysis. Akaike Information Criterion was used to determine the best-fitting model. This work is useful to predict the return periods of peak rainfall events during any month of the year that would occur in south-western Nigeria.\n\nThe authors have considered nine different probability plotting procedures and the best model out of these has been obtained. Four different survival models namely, exponential, log-logistic, Weibull, and log-normal are considered. The paper should be really helpful to understand how survival analysis can be used to predict the rainfall return period.\n\nIt would have been great if the authors could show plots or explain the distribution of the datasets. Also, they don't discuss if there are any missing observations or not. It is something that can be considered for further studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "129245", "date": "11 Apr 2022", "name": "Sohail Chand", "expertise": [ "Reviewer Expertise Statistical Modeling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors have fitted a survival model to rain return periods. They have considered nine different methods for the computation of the return period and four survival models. Here are a few suggestions for the improvement of the paper.\nA statistical summary of data should be provided. For this purpose, descriptive statistics and appropriate graphs can be used. Moreover, it will help readers to look at the statistical behavior of rain overall data.\n\nPrecisely define x1 and x2 predictors especially their types i.e. nominal or ordinal or scale. It is important to discuss how the categorical type variables are considered in the model and how their coefficients will be interpreted.\n\nGraphical presentation e.g. scatter diagrams can be helpful to visualize the relationship between response and predictors.\n\nModels' out-of-sample performance should be evaluated e.g. using some cross-validation techniques.\n\nIt would be worth considering, if possible, also some other performance measures in addition to Akaike Information Criterion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/11-83

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